Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness properties of ovarian cancer cells through reticulum stress and autophagy

Background Ovarian cancer has a high mortality rate mainly due to its resistance to currently used therapies. This resistance has been associated with the presence of cancer stem cells (CSCs), interactions with the microenvironment, and intratumoral heterogeneity. Therefore, the search for new therapeutic targets, particularly those targeting CSCs, is important for improving patient prognosis. HOOK1 has been found to be transcriptionally altered in a substantial percentage of ovarian tumors, but its role in tumor initiation and development is still not fully understood. Methods The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays. Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analysed. To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry. Immunofluorescence staining, Western-blotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer. Results In this study, we observed that reducing the levels of HOOK1 in ovarian cancer cells reduced in vitro growth and migration and prevented tumor formation in vivo. Furthermore, HOOK1 reduction led to a decrease in stem-like capabilities in these cells, which, however, did not seem related to the expression of genes traditionally associated with this phenotype. A proteome study, along with other analysis, showed that the downregulation of HOOK1 also induced an increase in endoplasmic reticulum stress levels in these cells. Finally, the decrease in stem-like properties observed in cells with downregulated HOOK1 could be explained by an increase in cell death in the CSC population within the culture due to endoplasmic reticulum stress by the unfolded protein response. Conclusion HOOK1 contributes to maintaining the tumorigenic and stemness properties of ovarian cancer cells by preserving protein homeostasis and could be considered an alternative therapeutic target, especially in combination with inducers of endoplasmic reticulum or proteotoxic stress such as proteasome inhibitors. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-024-03071-2.

While the findings offer valuable insights into the role of the Hook 1 gene across various biological processes and its poten�al as a therapeu�c target, a few drawbacks warrant clarifica�on and enhancement of rigor.
Thanks for your comments and valuable input to improve the manuscript.
1) Maintaining consistency in axis scales across histograms is crucial for accurately comparing data between different cell lines (e.g.panels in 1E; 1G; 2B; 2C; 3C; 3D, 4 E, 4F, ect.) and for facilita�ng a more reliable assessment of the differences or similari�es between experimental condi�ons.Therefore, it is highly recommended that the authors address this issue to improve the overall quality and accuracy of their study.
Whenever is possible we have modified the axis making them uniform to maintain consistency in axis scales across histograms.As suggested, we have changed them in all figures except the axes from OCT4 and KLF4 from Figure 3D and axes from Figure 6, that have not been changed to match between the two cell lines because their ranges are too different, and the results wouldn't be easily readable.The values of cell death in Figure 6 have not been relativized to establish the same axis between the two lines, as this would result in losing information about the predominant type of cell death in each scenario (apoptotic vs. non-apoptotic).
Also, IC50 values in Figure 4F have been normalized relative to the control to ensure axis uniformity between the two cell lines.Additional information regarding Figure 4F can be found in new Supplementary Figure 7.
2) To enhance the clarity and interpretability of figure 3A, it is essen�al for the authors to specify what the op�cal images represent, such as different treatment groups, control condi�ons, or specific experimental manipula�ons.
To improve the interpretability of the experiment as the reviewer suggests, Figure 3A   Addi�onally, the text in the manuscript has been altered to give more informa�on about where the concept of holoclones, meroclones and paraclones come from and what they represent.
Due to the previously found physiological alterations, we wondered whether HOOK1 might also play a role in the generation or maintenance of CSCs in ovarian tumors.For that purpose, we analyzed the proportion of different types of clones formed after seeding the cells at low density (REFs newly incorporated in the new manuscript) and performed a tumorsphere assay.In the analysis of the clones type, we observed a significant decrease in the percentage of holoclones (clones enriched in CSCs), and an increase in the percentage of paraclones (clones enriched in differentiated non-stem cells) under all conditions with reduced levels of HOOK1 (Figure 3A).(page 7-8, lanes 141-147) (REFs newly incorporated in the new manuscript) • Barrandon Y, Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci U S A. 1987 Apr;84(8):2302-6.• Li H, Chen X, Calhoun-Davis T, Claypool K, Tang DG. PC3 Human Prostate Carcinoma Cell Holoclones Contain Self-renewing Tumor-Initiating Cells. Cancer Res. 2008 Mar 15;68(6):1820-5.• Beaver CM, Ahmed A, Masters JR.Clonogenicity: holoclones and meroclones contain stem cells.Nie D, editor. PLoS One. 2014 Feb 26;9(2):e89834.
3) Is it biological relevant a 3%-8% cell death rate in figure 2B and 6.
Decreasing HOOK1 leads to an increase in cellular death, with rates ranging from doubling to quadrupling those observed in the parental cell lines (Figure 2B).This increase is sustained and persistent over time.Consequently, we anticipate that this sustained rise in cell death will significantly diminish the viability of ovarian cancer cells, which is biologically pertinent for tumor development and progression.Moreover, considering the suggested cause of this heightened mortality (stemming from an increase in ER stress), it is reasonable to infer that under conditions of elevated stress, such as in vivo tumor growth, there would be an even more pronounced increase in cell death due to decreased levels of HOOK1.Particularly if this effect is predominantly in CSCs, tumor growth would be impeded.This could elucidate why there is a lack of in vivo growth of cells with downregulated HOOK1 when injected into mice.
This conclusion is further supported by the heightened cell death induced by cell stressors.It is noteworthy that the increase in cell death significantly escalates when cells with downregulated HOOK1 are treated with other drugs inducing cellular stress, such as those activating the UPR (Figure 4F) or blocking autophagic flux (Figure 6B).Particularly in the latter case, we observe an increase in cell death reaching 15% in the OVCAR-8 line and 30% in the SKOV-3 line.Therefore, we believe that reducing HOOK1 could hold significant promise in the treatment of ovarian cancer patients, especially when combined with compounds that increase proteotoxic stress.

4)
In Figure 7A, the percentage of cell death among Epcam posi�ve SKOV-3 cells appears to exceed that of the total cell popula�on.This anomaly prompts ques�ons about the underlying biological mechanisms and experimental condi�ons.
Thanks for the comment.
The percentage of total alive EpCAM+ cells (Figure 7A, upper graph) is low due to the preferential death of these cells (Figure 7A, middle graph).The events are measured in percentage, so we can compare the effects, and perhaps this can give the clear difference observed.We think that this data supports the underlying hypothesis.
Even though the experiment has been repeated several times, the low abundance of EpCAM+ cells in clone 10 may pose challenges in accurately quantifying the percentage of dead cells under this condition.To avoid any doubt, we replicated this experiment using a different CRISPR clone of HOOK1 to validate our findings.We have incorporated the data of said clone in new Figure 7A.In said clone we also observe a decrease in EpCAM+ cells, accompanied by an elevation in cell death rates.In this case, the increase in cell death among EpCAM+ cells was less pronounced compared to the total population.This might explain the less pronounced decrease in EpCAM+ cells observed, as compared to clone 10.

Figure 7. Cell death induced by Hook1 downregulation may selectively impact CSCs. (A) Quantification by flow cytometry of the relative number of cells expressing the CSC marker EpCAM, EpCAM+ dead cells, and total dead cells in Hook1-downregulated cells. (B)
Quantification by flow cytometry of the relative number of parental cells expressing the CSC marker EpCAM, EpCAM+ dead cells, and total dead cells after the treatment with an ER stress inducer (Brefeldin A) or an autophagic flux blocker (chloroquine).The mean of 3 independent experiments ± SEM is represented.Statistical analysis was performed with Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001).The absence of an asterisk means that the data are not statistically significant.

5)
The absence of a legend in Figure 7 B severely impedes the comprehension of the results.Are reported the effects of inhibitors exclusively in wildtype cells?In summary, a thorough understanding of Figure 7's implica�ons necessitates access to its legend and addi�onal context regarding the experimental setup, cell types, treatments.Without such informa�on, a comprehensive interpreta�on remains elusive.
Thanks for calling our attention on this.There must have been some mistake on our part when incorporating the legend for Figure 7 in the text.We have included this legend and revised the to enhance clarity and have included it in its appropriate place within the text.The legend would be as follows:  The experimental setup is described in more detail in "Materials and methods".We have modified the section "Cell death assay in cells labeled with EpCAM by flow cytometry" to improve the description of the experimental design: The cells were maintained in culture with or without treatment for 48-72 hours (Table S1).Then, both suspended and adherent cells were collected.1x10 6 of those cells were resuspended in PBS with 2% FBS and 5 mM EDTA.Next, the cells were incubated with blocking agent (Miltenyi Biotec) for 10 minutes at 4 °C, and then, antibody labeling (Miltenyi Biotec) was performed for 15 minutes at room temperature and 15 minutes at 4 °C.Following this, two washes were performed using PBS with 2% FBS and 5 mM EDTA.After that, the previously described kit (Inmunostep) was used to label dead cells.Finally, the cells were examined using a FACSCanto II flow cytometer (BD Biosciences), and the results were analyzed using Diva software.

6)
In Figure 4B, it would be more appropriate to report the IC50 values on the curves rather than solely in the histogram, enhancing clarity and consistency.
We included histograms in Figure 4F to enhance result interpretation, as we believed their readability to be superior to that of the IC50 curves.However, to improve data consistency and clarity, as suggested, we have now incorporated the IC50 curves into new Supplementary Figure 7.We have also adjusted the values in Figure 4F relative to the parental cells to maintain consistency of the axes between the two cell lines, as previously recommended by the reviewer.Therefore, and to improve clarity, we have included in Supplementary Figure 7 a table with all the mean IC50 values +/-SEM for each condition:

Supplementary Figure 7. IC50 curves and mean values upon Hook1 downregulation. (A) IC50 curves of compounds that induce ER stress or inhibit proteasome function in Hook1downregulated cells. The mean of 3 independent experiments ± SEM is represented. (B) Table with mean values of IC50 ± SEM of cells with downregulated HOOK1 treated with ER stress
inducers or proteasome inhibitors.

7)
Moreover, discrepancies arise in the Western blot analysis of LC3B expression in SKOV cells between Figure 5 and supplemental Figure 8.Such inconsistencies undermine the reliability of the findings and raise concerns regarding experimental reproducibility and data integrity.Addressing these discrepancies and ensuring accurate figure labeling and presenta�on are essen�al for maintaining the scien�fic rigor and credibility of the study.
We believe that the discrepancies noted by the reviewer may stem from different exposition of blots due to the high increase of LC3B-II band of cells treated with chloroquine.

Figure 5A
Supplementary Figure 8A Figure 5A New Supplementary Figure 10A (previous Supplementary Figure 8A) In the SKOV-3 parental line under basal conditions (Figure 5A), the LC3B-II form is barely discernible in the WB, given the expectedly low levels of basal autophagy.In contrast, in the OVCAR-8 line, both forms of LC3B are distinctly observable in parental cells under basal conditions.In both lines, decreasing HOOK1 levels results in an increase in LC3B-II and the LC3B-II/LC3B-I ratio.However, in the SKOV-3 line, the LC3B-II band appears notably fainter, consistent with the baseline state of LC3B in the parental line.
When chloroquine is added to either of the cell lines (new Supplementary Figure 10A), as expected, there is a highly significant accumulation of LC3B-II, as its degradation is being inhibited.Consequently, we observe a substantial accumulation of LC3B-II across all conditions in both cell lines.Given that the baseline level of LC3B-II prior to chloroquine addition was higher in the OVCAR-8 line compared to SKOV-3, we are able to appreciate the LC3B-II band in untreated OVCAR-8 cells on the WB.However, in the SKOV-3 line, the significant LC3B-II accumulation upon chloroquine addition impedes observation of this band in untreated cells, as it would saturate the WB image exposure.Additionally, LC3B-I appears with considerably less intensity than in Figure 5A, again due to the significant accumulation of LC3B-II, that is masking the other form of LC3B.The only way to clearly observe LC3B in untreated cells in Supplementary Figure 10A would be to overexpose the WB membrane, thus losing LC3B information from the treated cells.Therefore, since in Supplementary Figure 10A we wanted to verify if chloroquine effectively blocked autophagic flux, we decided to use that WB image.

8)
The confusion arising from the authors' mislabeling of figures exacerbates the challenge of interpre�ng the findings accurately.Specifically, they misiden�fy supplemental figures, which may lead to misunderstanding or misinterpreta�on among readers.Addi�onally, in Figure 6, the incomplete labeling of the cell line name obstructs clear iden�fica�on.
We apologize for the confusing labelling of the figures and how it might have caused a misinterpretation of the content of the article.We have modified the labels of the Supplementary Figures and verified that all of the Figures are correctly cited in the text.We have also corrected the incomplete labeling of Figure 6.
Specifically, the authors must clarify whether Hook1 significantly influences cell death and cancer stem cell (CSC) death.The observa�on that stem cell genes are upregulated post-knockout suggests a complex rela�onship between Hook and CSC regula�on.To elucidate Hook's role, the authors should inves�gate the expression of stem cell genes in ovarian cancer cells with a dele�on of Hook1.This analysis would provide insights into how Hook1 dele�on impacts CSCrelated pathways and shed light on its therapeu�c poten�al.
We believe that the decrease in HOOK1 significantly affects the death of ovarian cancer cells.In Supplementary Figure 2A, we have incorporated the cell death rates of 5 additional CRISPR clones targeting HOOK1, further supporting our hypothesis.Notably, 4 of these newly introduced clones experience an increase in cell death.This increase is particularly notable in clones 3 of the OVCAR-8 line and 20 of the SKOV-3 line, where it is observed a tenfold increase in dead cells when compared to the parental cells.Regarding the death of CSCs specifically, we have added a new CRISPR clone to Figure 7A, providing more evidence that the decline in HOOK1 levels can specifically impact CSCs viability.
Regarding stem cell genes, to date, we have not found evidence that HOOK1 directly regulates the expression any of the genes related to stemness that we have examined (Figure 3C and 3D).
We believe that the increase in KLF4 and OCT4 that we observe in some cases might be derived from the increased ER stress (REF).However, we cannot discard that HOOK1 may be involved in the regulation of stem genes that have not been identified in this study.Nevertheless, beyond the regulation of these genes, we think that the decrease in CSC viability resulting from HOOK1 reduction could be the reason for the decline in stem properties that we have observed.In summary, while the findings regarding Hook1 are promising, the paper's inconsistencies and lack of clarity necessitate revisions to ensure its suitability for publica�on.Clarifying Hook's effects on cell death and CSC regula�on through addi�onal experiments would strengthen the manuscript and contribute to our understanding of its therapeu�c relevance in cancer.
Thanks for your comments.We hope that the new version clarifies these doubts.
Reviewer #2: The manuscript en�tled "Protein homeostasis maintained by HOOK1 levels promotes the tumorigenic and stemness proper�es of ovarian cancer cells through re�culum stress and autophagy", by Suarez-Mar�nez et al., inves�gates the role of the protein HOOK1 in ovarian cancer.
Since this tumor en�ty is notoriously difficult to treat, this work is of great clinical interest.HOOK1 has been described as linker between intracellular vesicles and molecular motors that run along microtubules.The authors found HOOK1 to be overexpressed in ovarian cancer, and its downregula�on impaired tumor cell prolifera�on in vitro and in vivo (s.c.injec�on in immunedeficient mice-but the actual number of experimental animals is indicated nowhere).These effects are atributed by the authors to a decreased tumor stemness upon downregula�on of HOOK1.Mechanis�cally, this is explained by increased ER stress and an ac�ve UPR response in cells with low HOOK1 levels, and altera�ons in the autophagic flux.
The authors present a nicely done study with an intriguing hypothesis, however, I do feel that some of their claims are not fully supported by the data.Whereas the tumorigenic proper�es of HOOK1 are clear and well documented by several orthogonal assays (except maybe for a model with forced expression that mimicks the higher HOOK1 levels seen in clinical samples), its effects on stemness are, at least in my view, not so convincing.Accordingly, the presumed influence on autophagic flux remains a bit vague, and no cell biological data are shown that would shed light on the involvement in kinesin/dynein-mediated vesicle transport, which could clear some of the central issues.
Thanks for your comments and valuable suggestions.

Specific points
Page 2: in the Abstract: why is it men�oned that Epcam was measured "only" by FACS?
It appears there was some English editing mistake during the Abstract submission process, as the content did not align with our intended message for the Methods section.We have rectified this mistake and present the revised version below: Methods.The downregulation of HOOK1 was performed in ovarian cancer cell lines using CRISPR/Cas9 technology, followed by growth in vitro and in vivo assays.Subsequently, migration (Boyden chamber), cell death (Western-Blot and flow cytometry) and stemness properties (clonal heterogeneity analysis, tumorspheres assay and flow cytometry) of the downregulated cell lines were analyzed.To gain insights into the specific mechanisms of action of HOOK1 in ovarian cancer, a proteomic analysis was performed, followed by Western-blot and cytotoxicity assays to confirm the results found within the mass spectrometry.Immunofluorescence staining, Westernblotting and flow cytometry were also employed to finish uncovering the role of HOOK1 in ovarian cancer.
(page 2, lanes 28-35) Page 5: the Crispr-KO clones show a "nearly complete" elimina�on of protein, but that means there is some remaining, residual expression of HOOK1 (maybe from escaping non-mutated cells that "contaminate" the cell cultures)?Has the mutated gene locus been sequenced?
The term "nearly complete elimination of protein" has been used because in the SKOV-3 line, we still observe residual levels of the HOOK1 protein by WB.However, in the OVCAR-8 line, no residual protein is observed (Figure 1D and Supplementary Figure 1).All clones used in the study have been sequenced to determine if they had undergone modifications at the desired locus.These sequencing results are depicted in Supplementary Figure 1B.In the case of the SKOV-3 line, we consider that residual protein levels may persist because the cells are heterozygous for the mutated allele.Furthermore, due to the CRISPR generation method and the low expression levels of HOOK1 that we have consistently observed in this line, we do not believe that any escape of non-mutated cells has occurred to contaminate our culture.
Recognizing that our previous phrasing in the text might lead to misunderstandings, we have revised that sentence in the manuscript to enhance clarity: The clones used for this study were selected among many by Western blot (WB) analysis (Supplementary Figure 1A) and sequenced (Supplementary Figure 1B) to confirm the mutation of the gene.In the OVCAR-8 cell line, a complete elimination of the protein was achieved, while in the SKOV-3 cell line, a very significant decrease in protein levels was observed.Additionally, in both cell lines, we observed very low residual mRNA levels compared to those of the parental line (Figure 1D).(page 6, lanes 113-118) Page 5: as men�oned above, the number of mice injected is not indicated We have included the number of mice injected per condition.It was 4. We have revised the "Xenograft in nude mice" section of the Methods to incorporate this information:
Page 6: please provide more details for the "clonability" assay -are the terms "paraclones" and "meroclones" etc. referring to the classical concept of Barrandon and Green (1987, I believe)?
The terms holoclones, meroclones, and paraclones in the clonal heterogeneity analysis indeed refer to the concepts initially described by Barrandon and Green in 1987.To facilitate understanding of this experiment, we have incorporated this reference into the manuscript text and updated the legend of Figure 3A.
Due to the previously found physiological alterations, we wondered whether HOOK1 might also play a role in the generation or maintenance of CSCs in ovarian tumors.For that purpose, we analyzed the proportion of different types of clones formed after seeding the cells at low density (REFs newly incorporated in the new manuscript) and performed a tumorsphere assay.In the analysis of the clones type, we observed a significant decrease in the percentage of holoclones (clones enriched in CSCs), and an increase in the percentage of paraclones (clones enriched in differentiated non-stem cells) under all conditions with reduced levels of HOOK1 (Figure 3A).Page 7: that is major issue: despite the fact that some proper�es of stemness (as assessed in vitro) seem to be regulated by HOOK1, the stem cell expression signature is not consistently altered (therefore, no effect on "classical" stem cell phenotype).This certainly is an interes�ng finding, but to claim that HOOK1 s�ll regulates stemness, but "likely through a variety of different mechanisms" is a bit bold.Is it really "stemness" that is induced by HOOK1 (as defined by tumor induc�on in an animal model by injec�on limi�ng dilu�ons of cells)?
Our findings suggest that HOOK1 plays a regulatory role in the physiology of CSCs, although not directly targeting the core Yamanaka genes.It is plausible that the reduction in HOOK1 could influence the expression of other stemness-related factors not addressed in our analysis.
Regardless of that, our observations indicate that diminished HOOK1 levels lead to increased ER stress and cell death, which we know specifically affects CSCs.We hypothesize that the reduction in viability of these cells could be the cause of the reduction in stem properties observed in our study, but without transcriptional alteration of stemnes genes.In the manuscript text, we have attempted to clarify that HOOK1 may be involved in the "maintenance" of CSCs, referring to their survival.However, recognizing the potential for ambiguity, we have revised the text to ensure clarity regarding the relationship between HOOK1 and stemness: Next, we analyzed the mRNA levels of genes commonly involved in the generation of CSCs due to their involvement in the dedifferentiation process.Surprisingly, we did not find a significant decrease in these genes when HOOK1 levels were reduced.In fact, in some cases, there was an increase, such as in KLF4 (Figure 3C).To confirm these results, we performed WB analysis and we observed that there was an increase in the amount of OCT4 and KLF4 proteins in the SKOV-3 cell line.However, we did not observe significant changes in any of these proteins in the OVCAR-8 line (Figure 3D).Therefore, the involvement of HOOK1 in diminishing stem properties in ovarian cancer cells seems to be independent of the expression of these genes conventionally linked to stemness, suggesting the presence of an alternative mechanism to account for such alterations.
(page 8, lanes 151-161) Along that line, Epcam is used as single marker to iden�fy cancer stem cells (Fig. 7), that is not really convincing, and other markers (e.g.CD44, CD24 etc.) should be included.
To explore alternative markers for identifying CSCs, we initially analyzed the expression of various markers proposed in the literature for ovarian cancer (REF1) in the cell lines used in our study (Supplementary Fig. 12A).Notably, CD44 and CD24 exhibit remarkably elevated expression levels in both cell lines.In fact, previous experiments conducted in our laboratory showed that 100% of the cells in both lines are positive for CD44 by flow cytometry, but not all reconstitute the culture.Therefore, we decided to exclude this marker from our analysis.Regarding CD24, there are studies in ovarian cancer suggesting that CSCs could be CD24+, while others suggest they could be CD44+/CD24-, similarly to breast CSCs (REF2, REF3).Given the 100% CD44+ cells in both cell lines, we considered selecting CD24-cells to study them as potential CSCs in our model.As for CD105 and CD10, we observed that they are expressed in the OVCAR-8 line, while in the SKOV-3 line, they have very reduced (neglectable) expression levels.On the other hand, CD117 is expressed in the SKOV-3 line but not in the OVCAR-8 line.Finally, CD106 is not expressed in either of the cell lines (Supplementary Fig. 12A).
Based on these data, we selected potential CSC markers alternative to EpCAM for the studied cell lines.Flow cytometry analysis was conducted to determine the percentage of positive cells for these markers in cells with reduced levels of HOOK1.According to the gene expression data from The Human Protein Atlas, we did not find CD117+ cells in the OVCAR-8 line, nor CD10+ or CD105+ cells in the SKOV-3 line (Supplementary Fig. 12A, 12B).Additionally, we found approximately 40% of CD24-cells in the OVCAR-8 line and 10% in the SKOV-3 line.This high percentage of CD24-cells suggests that the ability to maintain the culture was not dependent on this marker.Therefore, we decided to exclude it from our study.Based on the results obtained, we ultimately decided to select CD105 in the OVCAR-8 line and CD117 in the SKOV-3 line to continue their study as CSC markers (Supplementary Fig. 12B).
The flow cytometry analysis was conducted in triplicate for both cell lines using these markers.However, we found a significant variability in the percentage of cells positive for these markers across experiment replicates, especially in the CRISPR clones.Overall, we observed a positive correlation between cell passage number and the proportion of cells positive for these markers.
This increase was accompanied by a reduction in the proportion of dead cells positive for these markers (Supplementary Fig. 12C).Therefore, these results would suggest that ovarian CSC populations expressing these surface markers could be dynamic, changing over time, and becoming resistant to death caused by the reduction of HOOK1.The next point where the interpreta�on of the data is not en�rely consistent regards autophagy.The authors have tested two cell lines, and basically observed two opposing effects on autophagy (increase in OVCAR-8, and blockage in SKOV-3, Fig. 5).However, both cell lines show increased cell death upon downregula�on of HOOK1.This is explained by ER stress (OK), and by "par�al blockage of autophagy" (Discussion, p. 15), despite the fact that this only happens in one cell line, not the other.

Supplementary Figure 12. Study of CSC markers in the ovarian cancer cell lines OVCAR-8 and SKOV-3 with downregulated HOOK1. (A) Normalized gene expression values of reported CSC markers in OVCAR
Thanks for the constructive criticism.We replicated these experiments again, this time incorporating a larger set of HOOK1 CRISPR clones (5 clones in total in the OVCAR-8 line and 4 clones in the SKOV-3 line).These repetitions, coupled with the introduction of novel clones, has led us to clarify the hypothesis previously presented in the article.We observed that cells with reduced levels of HOOK1 exhibit an accumulation of autophagic vesicles, alongside a decrease in autophagosome-lysosome colocalization in the OVCAR-8 line, as we described also in the SKOV-3 line.These consistent alterations were observed across all 5 distinct clones analyzed (Figure 5B, Supplementary Figure 2C).However, the introduction of Brefeldin A to cells with diminished HOOK1 levels appeared capable of normalizing autophagosome-lysosome colocalization to that observed in parental cells treated with this drug.In light of our previous and recent experiments, we propose that autophagic flux may be partially impeded in both cell lines upon HOOK1 reduction.While this partial obstruction would occur in both lines, it appears more pronounced in the SKOV-3 line, as even the addition of Brefeldin A fails to induce an increase in autophagosome-lysosome colocalization reaching parental cell colocalization levels (Figure 2B).

Accordingly, we have modified the manuscript text to reflect our updated hypothesis:
To determine which of these two scenarios occurs in our model, we added brefeldin A to the cells to further increase ER stress (Figure S3A) and stimulate autophagy (Figure S3B) (Supp.Fig. 8) and analyzed autophagosome-lysosome fusion by immunofluorescence.In the OVCAR-8 cell line, we observed that the number, size, and fluorescence intensity of autophagosomes were similar in cells with reduced HOOK1 and parental cells.Furthermore, upon brefeldin A treatment, the size and number of autophagosomes did not change significantly under either condition.However, autophagosome-lysosome colocalization was significantly increased by the treatment, especially in HOOK1 CRISPRs, where it significantly surpassed what we observed in the parental cells (Figure 5B).These data suggest that cells with reduced HOOK1 levels in this cell line experience increased activation of autophagic flux to compensate for ER swelling.
Nevertheless, in the SKOV-3 cell line, We observed that cells with reduced HOOK1 levels, even without receiving any treatment, presented a higher number of autophagosomes than normal cells, and these vesicles were significantly larger than those observed in the parental cells.Furthermore, in the SKOV-3 cell line treated with the drug after treatment, the number, size, and fluorescence intensity of these vesicles increased even further, surpassing the levels found in the control cells.However, we found that autophagosome-lysosome colocalization was reduced in cells with downregulated HOOK1, especially in cell line SKOV-3, where the addition of Brefeldin A was not able to replicate the colocalization levels observed in the control cells (Figure 5B, Supp.Fig. 2C).upon treatment, autophagosome-lysosome colocalization significantly increased in the parental cells, while in the HOOK1 CRISPRs, the increase was not able to replicate the levels observed in the control cells (Figure 5B).This finding suggests that there is some blockade in the autophagic flux of the SKOV-3 cell line when HOOK1 is downregulated.This blockade would prevent the fusion of autophagosomes with lysosomes, leading to an accumulation of both structures.Hence, the reduction in HOOK1 affects autophagic flux; however, this phenomenon seems to be cell context dependent.Throughout -the uncropped immuno blot should be provided, since some of the blots are not exactly "high quality" We provide the uncropped immuno blots, that have been grouped in a PDF file called "Supplementary WB".
Fig. 8 the graphical abstract is a bit oversimplified and quite general, should either be revised or taken away In our study, we have observed that the reduction of HOOK1 affects general cellular processes that can impact cell viability.Therefore, as suggested by the reviewer, the graphical abstract may appear overly simplistic since we have not identified specific signaling pathways affected.We have revised the graphical abstract to include more information about the article's content and to enhance understanding of the discussion.However, if the reviewers still find it too general, we have no objection to its removal from the publication.

Reviewer #3:
The authors analyze the impact of HOOK1 expression in the maintenance of stemness proper�es of two ovarian cancer cell lines.Following knockdown of HOOK1 by CRISPR/cas9 edi�ng, they iden�fy an impairment of cell growth in vitro and in vivo.Analyzing the whole proteome, they also aim to demonstrate by func�onal assays an increased ER stress and autophagy which overall sustain an increased cell death.The study design and the experimental plan are appropriate but data descrip�on and interpreta�on should be improved.My major concerns are about the high variability observed at many different points (see also below) between the two cell lines and also among the different clones of the same cell line.
Although of poten�al interest, to correctly assess the biological relevance of the data shown, a higher number of clones should be considered at least in the key experiments showing ac�va�on of ER stress, autophagy and cell death.
Key experiments regarding the reviewer's suggested processes have been conducted on several different CRISPR clones of HOOK1 in two different cell lines.Specifically, randomly selected 3 and 2 CRISPR clones were employed in OVCAR-8 and SKOV-3 cell lines, respectively.The decrease of HOOK1 in these clones was validated by WB (Supplementary Fig. 2B).To verify the increase in ER stress caused by the downregulation of HOOK1 in ovarian cancer, WBs of key proteins involved in UPR activation were performed.In the OVCAR-8 line, an increase in ATF-4 and CHOP was observed in the 2 studied clones.Similarly, in the SKOV-3 line, an increase in GRP78 and CHOP was found in in one of the clones studied (Supplementary Fig. 2B).These findings suggest that, although with some variability among the clones, the decrease of HOOK1 is capable of inducing a general increase in ER stress in ovarian cancer cells.
Furthermore, flow cytometry analysis was employed to examine changes in cell death experienced by cells with downregulated HOOK1.It was found that 4 out of the 5 clones with reduced levels of HOOK1 studied on this occasion experience an increase in cell death.This increase is particularly notable in clones 3 of the OVCAR-8 line and 20 of the SKOV-3 line, where it is observed a tenfold increase in dead cells when compared to the parental cells (Supplementary Fig. 2A).
Lastly, the autophagic flux status of these HOOK1-deficient clones was studied by immunofluorescence, both in the absence and presence of the ER stress inducer Brefeldin A. We observed that parental cells exhibited small amount of autophagic vesicles stained with LC3B, which slightly increased upon treatment with Brefeldin A. However, we can appreciate that, in both cell lines, cells with reduced levels of HOOK1 clearly show an accumulation of autophagosomes, and these vesicles are much larger than those in parental cells.Additionally, we can see that, while parental cells treated with Brefeldin A undergo a marked increase in autophagosome-lysosome colocalization, the same does not occur in cells with just reduced levels of HOOK1 (Supplementary Fig. 2C).These results indicate that the increase in autophagic vesicles observed when downregulating HOOK1 cannot be solely explained by the increase in ER stress.Indeed, in some of the clones, we even observe a decrease in autophagosome-lysosome colocalization (Supplementary Fig. 2C).This fact, combined with the presence of an increased number and size of autophagosomes, would again suggest that there may be some kind of blockage of autophagic flux when reducing HOOK1, leading to the accumulation of these vesicles.
Upon treatment with Brefeldin A, CRISPR clones with downregulated HOOK1 show an increase in the autophagosome-lysosome colocalization similar to that found in control cells (Supplementary Fig. 2C).Therefore, although autophagic flux may be partially blocked, it is not completely inactive.This observation aligns with the dependency of cells with reduced levels of HOOK1 on autophagic flux, as evidenced by the pronounced increase in mortality of these cells observed upon chloroquine treatment (Figure 6B).
In summary, we could conclude that the different clones with reduced levels of HOOK1 analyzed (5 in total in the OVCAR-8 line and 4 in total in the SKOV-3 line) behave similarly to each other and in both cell lines.

Supplementary Figure 2. Analysis of cell death, ER stress and autophagy in different HOOK1 CRISPR clones in OVCAR-8 and SKOV-3 cell lines. (A) Quantification by flow cytometry of the relative number of dead cells when Hook1 is downregulated. (B) Protein levels of UPR-associated proteins in Hook1-downregulated cells. (C) Immunofluorescence staining of LC3B (green) and LAMP2 (red) proteins in Hook1-downregulated cells treated with brefeldin A. DAPI (blue) was
used as a nuclear stain, and the merge of the 3 markers is shown.The autophagosome size, number and relative fluorescence intensity, as well as the colocalization index of autophagosomes-lysosomes, were measured using ImageJ software.
Below is a point by point lis�ng of the other major issues *In the Introduc�on lines 85-88 are not clear.Data available on HOOK1 expression/func�on in cancers should be beter described with more insights and details.
A more detailed information about HOOK1 proposed role in cancer have been included in the Introduction.Some additional changes have been made in the Introduction in order to make it more concise and focused on the relation between HOOK1 and cancer.
The role of HOOK1 in cancer has not been explored in depth.However, a few studies have linked this protein to different types of tumors.For instance, a decrease in HOOK1 has been observed in hepatocellular carcinoma, where its downregulation has been associated with increased malignancy (REF1).Furthermore, HOOK1 has been proposed as a biomarker in papillary mucinous neoplasm following a proteomic screening (REF2).Nonetheless, the mechanism by which HOOK1 may be involved in cancer remains unclear.The most studied hypothesis in this regard is the association between HOOK1 and the phosphatase SHP2, implicated in tumorigenesis and metastasis in different types of tumors (REF3, REF4, REF5).However, the function of SHP2 as an oncogene or tumor suppressor gene is controversial and apparently depends on the tumor type (REF4).It has been described that HOOK1 is capable of binding to SHP2, and it has been suggested that it acts as an inhibitor of its phosphatase activity (REF4).In lung cancer cells, it was observed that while SHP2 positively regulates TEM, HOOK1 has the opposite effect (REF4).Nonetheless, other studies have linked HOOK1 to the progression of different types of tumors independently to its proposed interaction with SHP2 (REF1, REF6, REF7).Hence, the precise role of HOOK1 in cancer and its underlying mechanism remain unclear.( 1. Descrip�on of HOOK1 altera�on is not clear.Specifically, is not clear if the % of altera�on frequency is referred to the total varia�ons types or the total of case analyzed.Also, the rela�ve percentages shown are not consistent with KM curves reported in figure 1C as the total of altered case is really few.
We have answered questions 1 and 2 together.1C key details need to be added: for each cohort it must be shown the number of cases and the number of events.However, since the two groups appear to be extremely unbalanced with the altered group having so few events, it would not appropriate to perform this type of analysis.

In Figure
The 3.5% alteration frequency refers to all alterations found in the analyzed cases.Specifically, the percentage of amplifications in the total cases is 2.23%.In the Kaplan-Meier analysis 18 individuals compose the group with alterations and 552 individuals compose the group without alterations.Therefore, the percentage shown in the graph of alterations is consistent with the data presented in the Kaplan-Meier analysis.However, as highlighted by the reviewer, the size of the group with alterations is quite reduced, so probably it is necessary to increase the group with HOOK1 alterations to extract more robust conclusions.Therefore, we have analyzed by mRNA expression.See below.
3. Since the authors explored both GEPIA and cBIO portal I would consider the prognos�c impact of HOOK1 mRNA expression rather than gene altera�on, also considering their working hypothesis and the experiments that they performed in vitro (CRISPR knockdown) Thank you for the suggestion.We agree with the reviewer that a Kaplan-Meier analysis separating ovarian cancer patients by levels of HOOK1 expression would better align with the content of the article compared to grouping by alterations in HOOK1.Considering also the limited number of patients in the group with alterations in the previous analysis, we have replaced Figure 1C with a new plot displaying overall patient survival stratified by HOOK1 expression levels.We have also swapped the positions of Figures 1B and 1C to maintain a logical order in the text.

Consequently, Figure 1C now becomes the new Figure 1B. The manuscript text has been updated to incorporate the new information:
In previous studies, HOOK1 has been suggested to be linked to platinum resistance in ovarian tumors 24 .To undercover its significance in this type of cancer, firstly, we studied it in multiple databases.Using the online database GEPIA, We observed that HOOK1 expression was significantly higher in ovarian tumors than in normal tissue (Figure 1A) and that the overall survival tends to be worse in tumors where this gene is highly expressed (Figure 1B).In addition, in the cBioPortal database, we found that there was a high percentage of ovarian tumors with amplifications in this gene (Figure 1C).Altogether, these data show that there is a high percentage of ovarian tumors with HOOK1 alterations and expression changes and that these alterations modifications may be associated with a worse prognosis in patients.

Please add a reference for the GEPIA database.
A new section called "Public database analysis" has been incorporated to the Methods.In this section we include the references of all the databases used in this study.
(page 25, lanes 535-539) *Flow citometry assay to detect apoptosis and related data interpreta�on should be beter described in both Materials and Methods and results sec�on.Given the variability and in some cases the cells-specificity observed, at least in supplementary materials a representa�ve FACS should be shown.
We have improved the explanation of the flow cytometry conducted to detect apoptosis and the presence of CSC markers.The "Materials and Methods" section has been modified to better explain these experiments, and representative images of the flow cytometry charts have been included in Supplementary Figure 11.The text of the "Results" section has also been modified to enhance the interpretability of the experiments.Modifications in the text are underlined.

Cell death assay
The cells were maintained in culture with or without treatment for 48-72 hours (Table S1).Then, both suspended cells and adherent cells were collected, and the 'Apoptosis Detection' kit (Immunostep) was used to measure cell death following the manufacturer's instructions.Briefly, cells were stained with Annexin V for 15 minutes at room temperature and in darkness.Subsequently, they were washed with Binding Buffer solution and centrifuged to remove the supernatant.Finally, they were incubated with propidium iodide for 5 minutes at room temperature.A FACSCanto II flow cytometer (BD Biosciences) was employed to detect the staining, and the results were analyzed using Diva software.Cells stained only with Annexin V were considered in early apoptosis, cells stained only with propidium iodide were considered in necrosis, and cells stained with both markers were considered in late apoptosis.All cells stained with propidium iodide were considered dead (Supp.Fig. 11).

Cell death assay in cells labelled with EpCAM by flow cytometry
The cells were maintained in culture with or without treatment for 48-72 hours (Table S1).Then, both suspended and adherent cells were collected.1x10 6 of those cells were resuspended in PBS with 2% FBS and 5 mM EDTA.Next, the cells were incubated with blocking agent (Miltenyi Biotec) for 10 minutes at 4 °C, and then, antibody labeling (Miltenyi Biotec) was performed for 15 minutes at room temperature and 15 minutes at 4 °C.Following this, two washes were performed using PBS with 2% FBS and 5 mM EDTA.After that, the previously described kit (Inmunostep) was used to label dead cells.Finally, the cells were examined using a FACSCanto II flow cytometer (BD Biosciences), and the results were analyzed using Diva software.The number of dead cells was measured in EpCAM+ cells and the whole population.All cells stained with propidium iodide were considered dead.

HOOK1 affects the tumorigenic properties of ovarian cancer cells in vitro and impairs their ability to form tumors in vivo
[…] For this purpose, we performed a flow cytometry assay using Annexin-V and a DNA intercalating agent.We observed that cells with reduced levels of HOOK1 showed an increase in the percentage of dead cells, both through apoptotic and nonapoptotic cell death (Figure 2B, Supp.Fig. 2A, Supp.Fig. 11).Then, we investigated the alteration of proteins related to apoptotic pathways, such as cleaved CASP3, CASP9, and PARP, and p-H2AX.Through WB analysis, we found an increase in the quantity of all the studied markers (Figure 2C).Thus, it appears that the reduction in HOOK1 increases the activation of the apoptotic pathway in ovarian cancer cells and leads to an increase in cell death, both through apoptosis-dependent and apoptosis-independent mechanisms.[…] (page 7, lanes 128-136)

Cell death induced by Hook1 downregulation is dependent on the ER stress response and autophagic flux.
[…] As previously demonstrated, the reduction in HOOK1 levels is capable of activating apoptotic signaling pathways and causing an increase in cell death (Figure 4B,4C).Therefore, we investigated whether the observed activation of the UPR in our model is responsible for this phenomenon.For that purpose, we used the drug tauroursodeoxycholic acid (TUDCA) to reduce ER stress levels (128).First, we confirmed that the drug effectively reduced ER stress in our model (Figure S4).Next, we studied cell death by flow cytometry and performed a WB of apoptosisrelated markers.In the OVCAR-8 cell line, we observed that apoptotic markers did not change significantly in clone 19, while in clone 5, we observed an increase in cleaved PARP and p-H2AX.By flow cytometry, we found that TUDCA seemed to reduce cell death in clone 5; however, in clone 19, the changes in cell death were not as clear (Figure 6A).In the SKOV-3 cell line, there was a reduction in cleaved CASP3, CASP9, and PARP in CRISPR clone 10 but not in clone 3. Flow cytometry analysis showed that in both CRISPR clones, adding TUDCA to the medium reduced cell death.However, clone 10 exhibited a decrease in early-stage apoptosis, while clone 3 showed a reduction in necrosis, which may explain why we did not observe changes in apoptotic markers in this latter clone (Figure 6A).These results suggest that the inhibition of ER stress attenuates cell death in cells with reduced HOOK1 levels, although it does not completely rescue the phenotype.
As previously demonstrated, reducing HOOK1 levels activates apoptotic signaling pathways, leading to increased cell death (Figure 4B, 4C).To investigate whether the activation of the UPR is responsible of this death, we used tauroursodeoxycholic acid (TUDCA) to reduce ER stress levels.Although TUDCA effectively reduced ER stress (Supplementary Fig. 9), its impact on cell death shows some clonal variability.In OVCAR-8 cells, apoptotic markers showed no significant changes, and TUDCA seemed to reduce cell death in clone 5 but had unclear effects in clone 19.In SKOV-3 cells, TUDCA reduced cleaved CASP3 and CASP9 and flow cytometry revealed reduced cell death in both clones, with clone 10 showing primarily a decrease in early-stage apoptosis and clone 3 displaying reduced necrosis (Figure 6A).Altogether, these findings suggest that inhibiting ER stress attenuates cell death in cells with reduced HOOK1 levels, albeit not completely rescuing the phenotype.
In contrast, activation of autophagic flux was reported to serve as a survival mechanism in cells with high ER stress.However, we found an impeded autophagic flux when we decreased HOOK1 levels (Figure 5B, Supp.Fig. 2C).Therefore, we considered it important in order to determine whether autophagy contributes protectively or inductively to cell death in our model.For this purpose, we used chloroquine as an inhibitor of autophagic flux [32].As previously described, We first confirmed its effectiveness by WB and immunofluorescence analyses (Figure S5) (Supp.Fig. 10).Subsequently, we observed that when we added chloroquine to cells with reduced HOOK1 levels, some apoptotic markers, such as CASP3, CASP9 and PARP, were reduced in both cell lines (Figure 6B).However, by flow cytometry, we observed an increase in the percentage of dead cells after the treatment, which was significantly more pronounced in the cells with reduced HOOK1 levels.This increase was not due to the activation of apoptosis; instead, we found a very significant increase in the number of necrotic cells (Figure 6B).Therefore, these data suggest that cells with reduced HOOK1 levels are more sensitive to the blockage of autophagic flux, which likely acts as a protective mechanism to restore cellular proteostasis.Furthermore, when autophagy is blocked, the preferred route for cell death in HOOK1-depleted cells is apoptosisindependent.This result, coupled with the increase of vesicle colocalization in CRIPSR clones following the addition of brefeldin A (Figure 5B, Supp.Fig. 2C), indicates that autophagic flux is not completely blocked in cells with reduced levels of HOOK1, retaining some degree of functionality.

CSCs might be particularly affected by the cell death induced by Hook1 downregulation
Our data provide evidence that reducing HOOK1 leads to a decrease in cancer stem-like properties in ovarian cancer cells.However, this phenomenon was not dependent on genes traditionally associated with dedifferentiation.Therefore, we studied whether there could be a relationship between the reduction in stem-like capabilities and the occurrence of ER stressassociated cell death when HOOK1 was reduced.To investigate this, we analyzed the percentage of dead cells positive for EpCAM, a CSC marker.In both ovarian cancer cell lines, we observed that downregulating HOOK1 caused a decrease in the percentage of EpCAM-positive cells compared to that of parental cells.This decrease occurred in all CRISPR clones but was particularly striking in clone 10 of the SKOV-3 cell line.In this clone, we found an approximately 10-fold increase in cell death compared to that of the parental line.In all other clones, the reduction in HOOK1 levels increased the death rate of EpCAM-positive cells by approximately 2fold (Figure 7A).The analysis of cell death specifically in EpCAM+ cells revealed an approximately 2-fold increase in cell death in most HOOK1 CRISPRs in comparison to parental cells.Strikingly, clone 10 of SKOV-3 cell line showed a 10-fold increase in cell death compared to that of the parental line.This result correlates with the very reduced percentage of EpCAM+ cells that we found in said clone (Figure 7A).These data support the hypothesis that the reduction in HOOK1 leads to a preferential decrease in CSCs in ovarian cancer due to an increase in their cell death, probably due to unsustainable ER stress.[…] (page 13, lines 262-274) *The authors did not observe varia�ons in stem marker expression upon HOOK1 downregula�on.These data should be beter discussed.Would it be possible that HOOK1 acts downstream of the yamanaka factors?It would be interes�ng to evaluate if impairment of one on these factors may impact on HOOK1 expression.
The point raised by the reviewer is truly intriguing.In this study, our focus has been on elucidating the effects of HOOK1 on ovarian cancer cells rather than exploring its regulatory mechanisms.However, as the author suggests, it is possible that HOOK1 is downstream of one of the Yamanaka factors, which would be interesting to explore for future research.To date, no investigation has probed the interplay between Yamanaka factors and HOOK1.Due to the lack of time to conduct a more in-depth study, but given the interest generated by the question, we have performed a basic bioinformatic analysis using publicly available data.
To investigate the potential binding of Yamanaka factors to the promoter region of HOOK1, we extracted a 1 kb sequence upstream of the gene's transcription start site from the latest human genome assembly (hg38) using the UCSC Genome Browser (http://genome.ucsc.edu/index.html).This sequence was then queried against the Jaspar database (http://jaspar.binf.ku.dk/) to identify putative transcription factor binding motifs, specifically for SOX2, OCT4, KLF4, and C-MYC.While some possible binding motifs were detected for SOX2 and OCT4, KLF4 emerged as the transcription factor with the most abundant and highest-scoring motifs.Consequently, our analysis suggests that if any Yamanaka factor were to interact with the HOOK1 promoter, KLF4 would be the principal candidate.Nevertheless, the expression of HOOK1 may also be subject to modulation by Yamanaka factors, even in the absence of direct regulation of its promoter.To explore this possibility further, we analyzed RNA-seq data from OVCAR-3 ovarian cancer cells reprogrammed with the four Yamanaka factors.We observed a decrease in HOOK1 expression levels in the reprogrammed cell line compared to the parental counterpart (REF).This result suggests that, while Yamanaka factors may not directly induce HOOK1 expression, they could exert an indirect influence.Username: reviewer_pxd050782@ebi.ac.ukPassword: GnLXzadr

SOX2
The section "Materials and Methods" has been updated to include this information and the citation of the software used to analyse the data:

Proteomic analysis
[…] Protein identification and label-free quantitation.MS/MS spectra were searched against the reference proteome FASTA file (42161 entries; swissprot_2017_03_human_canonical_and_isoform).Enzyme specificity was set to trypsin, and up to two missed cleavages were allowed.Cysteine carboxamidomethylation (Cys, +57.021464Da) was treated as fixed modification and methionine oxidation (Met, +15.994915Da) and Nterminal acetylation (N-terminal, +42.010565Da) as variable modifications.Peptide precursor ions were searched with a maximum mass deviation of 4.5 ppm and fragment ions with a maximum mass deviation of 20 ppm.Peptide and protein identifications were filtered at an FDR of 1% using the decoy database strategy.The minimal peptide length was seven amino acids.Proteins that could not be differentiated based on MS/MS spectra alone were grouped into protein groups (default MaxQuant settings).Searches were performed with the label-free quantification option selected.Proteins were quantified by spectral counting, that is, the number of identified MS/MS spectra for a given protein (Liu et al., 2004) combining the five fractions per sample.Raw counts were normalized on the sum of spectral counts for all identified proteins in a particular sample, relative to the average sample sum determined with all samples.To find statistically significant differences in normalized counts between sample groups, we applied the beta-binomial test (Pham et al., 2010), which takes into account within-sample and betweensample variation using an alpha level of 0.05.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD050782.The generated data were filtered using the R platform [2] to obtain proteins whose expression increased (FC > 1) or decreased (FC < -1) significantly (p < 0.05) and analyzed using the ShinyGO 0.77 [3]  [2] R Core Team (2021).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.URL https://www.R-project.org/.
[3] Steven Xijin Ge, Dongmin Jung, Runan Yao, ShinyGO: a graphical gene-set enrichment tool for animals and plants, Bioinformatics, Volume 36, Issue 8, April 2020, Pages 2628-2629, https://doi.org/10.1093/bioinformatics/btz931 [4] Szklarczyk D, Franceschini A, Wyder S, et al.STRING v10: protein-protein interac�on networks, integrated over the tree of life.Nucleic Acids Res.2015;43(Database issue):D447-D452.doi:10.1093/nar/gku1003*Given that the DE proteome is consistently different among the two cell lines with very few commonly modulated proteins, it is not clearly explained the ra�onale of choice to analyze and show the expression of proteins reported in Figure 4E, which apparently are not among those commonly DE and have a high variable trend among clones also within a single cell line.Since this high variability, a true valida�on of the DE protein should be performed before exploring their possible func�onal impact.
There is variability in the differentially expressed proteins between the two cell lines, although certain proteins exhibit similar alterations in both, as demonstrated in Supplementary Figure 6C  and D. In fact, one of the proteins appearing in Supp.Fig. 6C (HSPA5/GRP78) has been analyzed by WB in Figure 4E to confirm the results of the proteomics.However, it is worth noting that in both cell lines, we found an alteration in the expression of proteins involved in protein folding in the ER.This prompted us to investigate whether there was an increase in ER stress due to an accumulation of misfolded proteins.To this end, we analyzed the expression of different proteins belonging to the ATF6 and PERK pathways, both activated in response to ER protein accumulation.We also assessed the sensitivity of cells with HOOK1 reduction to various ER stressinducing compounds to confirm these results and ensure the increase in ER stress.
We have not continued to explore other potentially dysregulated proteins from the proteomic analysis associated to different processes, although it would certainly be of interest for future research.
*Please improve the clarity of the descrip�on of figure 5, adding also some quan�ta�ve data to help to interpret the possible effect of the different treatments.
We have assumed that the clarity issue arises in new Figure 6, which involves various treatments without accompanying WB quantification.We provide the quantification data for these WB analyses conducted on cells treated with TUDCA and chloroquine.Additionally, we have modified the manuscript text relating to this figure to improve readability and comprehension of the results.[…] As previously demonstrated, the reduction in HOOK1 levels is capable of activating apoptotic signaling pathways and causing an increase in cell death (Figure 4B,4C).Therefore, we investigated whether the observed activation of the UPR in our model is responsible for this phenomenon.For that purpose, we used the drug tauroursodeoxycholic acid (TUDCA) to reduce ER stress levels (128).First, we confirmed that the drug effectively reduced ER stress in our model (Figure S4).Next, we studied cell death by flow cytometry and performed a WB of apoptosisrelated markers.In the OVCAR-8 cell line, we observed that apoptotic markers did not change significantly in clone 19, while in clone 5, we observed an increase in cleaved PARP and p-H2AX.By flow cytometry, we found that TUDCA seemed to reduce cell death in clone 5; however, in clone 19, the changes in cell death were not as clear (Figure 6A).In the SKOV-3 cell line, there was a reduction in cleaved CASP3, CASP9, and PARP in CRISPR clone 10 but not in clone 3. Flow cytometry analysis showed that in both CRISPR clones, adding TUDCA to the medium reduced cell death.However, clone 10 exhibited a decrease in early-stage apoptosis, while clone 3 showed a reduction in necrosis, which may explain why we did not observe changes in apoptotic markers in this latter clone (Figure 6A).These results suggest that the inhibition of ER stress attenuates cell death in cells with reduced HOOK1 levels, although it does not completely rescue the phenotype.
As previously demonstrated, reducing HOOK1 levels activates apoptotic signaling pathways, leading to increased cell death (Figure 4B, 4C).To investigate whether the activation of the UPR is responsible of this death, we used tauroursodeoxycholic acid (TUDCA) to reduce (ER) stress levels.Although TUDCA effectively reduced ER stress (Figure S4), its impact on cell death varied.In OVCAR-8 cells, apoptotic markers showed no significant changes, and TUDCA seemed to reduce cell death in clone 5 but had unclear effects in clone 19.In SKOV-3 cells, TUDCA reduced cleaved CASP3 and CASP9 and flow cytometry revealed reduced cell death in both clones, with clone 10 showing primarily a decrease in early-stage apoptosis and clone 3 displaying reduced necrosis (Figure 6A).Altogether, these findings suggest that inhibiting ER stress attenuates cell death in cells with reduced HOOK1 levels, albeit not completely rescuing the phenotype.
In contrast, activation of autophagic flux was reported to serve as a survival mechanism in cells with high ER stress.However, we found an impeded autophagic flux when we decreased HOOK1 levels (Figure 5B, Supp.Fig. 2C).Therefore, we considered it important in order to determine whether autophagy contributes protectively or inductively to cell death in our model.For this purpose, we used chloroquine as an inhibitor of autophagic flux [32].As previously described, We first confirmed its effectiveness by WB and immunofluorescence analyses (Figure S5) (Supp.Fig. 10).Subsequently, we observed that when we added chloroquine to cells with reduced HOOK1 levels, some apoptotic markers, such as CASP3, CASP9 and PARP, were reduced in both cell lines (Figure 6B).However, by flow cytometry, we observed an increase in the percentage of dead cells after the treatment, which was significantly more pronounced in the cells with reduced HOOK1 levels.This increase was not due to the activation of apoptosis; instead, we found a very significant increase in the number of necrotic cells (Figure 6B).Therefore, these data suggest that cells with reduced HOOK1 levels are more sensitive to the blockage of autophagic flux, which likely acts as a protective mechanism to restore cellular proteostasis.Furthermore, when autophagy is blocked, the preferred route for cell death in HOOK1-depleted cells is apoptosisindependent.This result, coupled with the increase of vesicle colocalization in CRIPSR clones following the addition of brefeldin A (Figure 5B, Supp.Fig. 2C), indicates that autophagic flux is not completely blocked in cells with reduced levels of HOOK1, retaining some degree of functionality.(page 11-12, lines 233-260) *Line 252 please clarify the statement about 'altera�on in the autophagic flux' In line 252, the mention of alterations in the autophagic flux specifically refers to the variations observed in autophagy discussed in the preceding section ("4.Hook1 downregulation causes changes in autophagic flux that are cell context dependent.").We have revised the sentence in line 252 to offer clearer context.(See previous answer).*Discussion should be revised.It has not to be a mere summary of the results obtained.Figures' cita�on should be avoided and cri�cal discussion has to be enhanced.
The discussion has been thoroughly revised to enhance its comprehensibility and cri�cal component, as well as to avoid referencing figures from the results.Below are the changes made to the text:

Discussion
The downregula�on of HOOK1 in ovarian cancer cell lines has resulted in a decrease in their tumorigenic and stemness proper�es while significantly increasing cell death.However, we have not found altera�ons in the expression of genes tradi�onally related to the stem cell phenotype that could explain this phenomenon.In some cases, we even found an increase of the expression of those genes, as in the case of KLF4.This could be explained by the increased ER stress observed when HOOK1 levels decrease.A recent study linked increased ER stress to the induc�on of KLF4 expression in melanoma cells, as an adap�ve mechanism inhibi�ng UPRinduced apoptosis and promo�ng metastasis [33].Furthermore, other studies have observed that the increase in GRP78 can promote the expression of OCT4 in head and neck and breast cancer cells [34,35].Hence, it is worth considering that the increase in ER stress may account for the lack of reduc�on in these genes.HOOK1 was found to be overexpressed in ovarian cancer tumors (Figure 1A), and its downregula�on in cell lines of this tumor resulted in a decrease in cell prolifera�ve capacity (Figure 1C, 1D) and migra�on (Figure 2A) while significantly increasing cell death (Figure 2B).Moreover, xenogra�ing these cells into mice failed to form tumors (Figure 1E).These data suggest a clear role for HOOK1 in the genera�on or maintenance of ovarian tumors.Addi�onally, some proper�es related to CSCs were reduced when HOOK1 levels decreased (Figure 3A, 3B).However, we did not observe changes in genes tradi�onally related to the stem cell phenotype.In some cases, we found an increase in some genes, such as KLF4 (Figure 3C, 3D).Although this increase seems to contradict the decrease in CSC proper�es, it could be explained by the increased endoplasmic re�culum (ER) stress observed when HOOK1 levels decrease (Figure 4).A recent study linked increased ER stress to the induc�on of KLF4 expression in melanoma cells.KLF4 expression acts as an adap�ve mechanism against ER stress, inhibi�ng UPR-induced apoptosis and promo�ng metastasis 25 .
Tumor cells commonly experience ER stress due to various stress sources [36].While no established link between HOOK1 and ER stress exists, we can speculate on poten�al causes.The occurrence of ER stress is common in tumor cells due to the numerous sources of stress that they experience 26 .To date, no rela�onship has been described between the HOOK1 protein and the occurrence of ER stress.However, we can speculate about the possible causes of this phenomenon.The protein HOOK2, from the same family as HOOK1, is involved in the forma�on and maintenance of aggresomes [37], structures whose inhibi�on has been associated with accumula�on of misfolded proteins and ER stress [38,39].HOOK1 might play a similar role in the forma�on of this structure, possibly through the transport of HOOK1 could be involved in transpor�ng misfolded proteins to this structure due to its role in retrograde protein transport.Notably, there are structural and func�onal similari�es between the HDAC6 protein, which is essen�al for aggresome forma�on, and the HOOK protein family [11,38].Addi�onally, the FHF complex (HOOK, FTS, and FHIP) has been associated with the mo�lity of tubular intermediaries that allow ER transport to the Golgi apparatus 14 .Hindered transport between these two organelles could also lead to increased ER stress.
In addi�on to UPR ac�va�on, cells with reduced HOOK1 levels show changes in autophagic flux (Figure 5).In recent years, a strong interconnec�on between the UPR and autophagy has been described 29 .The role of autophagy in cancer is complex and context dependent.Its func�on in maintaining homeostasis plays an important role in preven�ng tumorigenesis.However, autophagic induc�on in tumors exposed to hypoxia and/or nutrient depriva�on can provide the necessary energy to promote cell survival and chemoresistance 26 .In contrast, excessive autophagic ac�va�on can also trigger cell death through different mechanisms 30 .Therefore, the exact role of autophagy in cancer is dependent on tumor type, stage, and tumor microenvironmental condi�ons.In cells with reduced HOOK1 levels, UPR ac�va�on would be expected to increase autophagic flux.However, the results suggest that in the OVCAR-8 cell line, decreased HOOK1 levels indeed lead to an increase in autophagy, while in the SKOV-3 cell line, HOOK1 reduc�on seems to cause some kind of blockage in autophagic flux (Figure 5).This blockage would prevent autophagosome-lysosome fusion, leading to an accumula�on of these structures.
This par�al blockage of autophagy could be related to the interac�on of the FHF complex, including HOOK proteins, with the HOPS complex.When one of the complex proteins is eliminated, the ability of Vps18 to promote lysosome clustering is compromised.These data suggest that the FHF complex may help coordinate the movement or interac�on of vesicles through the HOPS complex 13 .The HOPS complex is involved in the fusion of late endosomes and autophagosomes with lysosomes in mul�ple organisms.Therefore, the loss of any of its subunits leads to the accumula�on of late endosomes and autophagosomes 31-33 .Thus, it is possible that HOOK1 loss blocks autophagic flux by interfering with the proper func�oning of the HOPS complex.Therefore, studying the interac�on of HOOK1 with the HOPS complex in the future could help elucidate why autophagic flux might be blocked in our model and why it seems to affect the two studied cell lines differently.
Independently from its origin, Both the increase in increased ER stress and the par�al blockage of autophagic flux could be responsible for the increase eleva�on in cell death observed in cells with downregulated HOOK1 (Figure 2B).Sustained ER stress is cytotoxic to cells, leading to the ac�va�on of the apopto�c pathway and other cell death mechanisms [40,41].We found that reducing ER stress in our model par�ally reverses cell death, indica�ng that ER stress may be responsible, at least in part, for this death.To corroborate the existence of a cytotoxic effect of ER stress in our model, we used TUDCA to reduce it.We found that the use of TUDCA par�ally reverses cell death, indica�ng that ER stress may be responsible, at least in part, for this death.However, we did not observe a complete reversal of the phenotype when adding the drug (Figure 6A).This could be explained because the inhibi�on of ER stress was not sufficiently effec�ve, although we did observe a reduc�on in ER stress markers when using TUDCA (Figure S4).Therefore, it is possible that cells with reduced HOOK1 levels experience increased cell death due to more than one factor.
In addi�on to UPR ac�va�on, cells with reduced HOOK1 levels show changes in autophagic flux.In recent years, a strong interconnec�on between the UPR and autophagy has been described [31].The role of autophagy in cancer is complex and context dependent.Its func�on in maintaining homeostasis plays an important role in preven�ng tumorigenesis.However, autophagic induc�on in tumors exposed to hypoxia and/or nutrient depriva�on can provide the necessary energy to promote cell survival and chemoresistance [36].In contrast, excessive autophagic ac�va�on can also trigger cell death through different mechanisms [42].Therefore, the exact role of autophagy in cancer is dependent on tumor type, stage, and tumor microenvironmental condi�ons.In cells with reduced HOOK1 levels, UPR ac�va�on would be expected to increase autophagic flux.However, our results suggest that the decrease in HOOK1 could cause some kind of blockage in this process, that would prevent autophagosome-lysosome fusion, leading to an accumula�on of these structures.This par�al blockage of autophagy could be related to the interac�on of the FHF complex, including HOOK proteins, with the HOPS complex.It has been suggested that the FHF complex may help coordinate the movement or interac�on of vesicles through the HOPS complex [13], which is involved in the fusion of late endosomes and autophagosomes with lysosomes in mul�ple organisms.Therefore, the loss of any of its subunits leads to the accumula�on of late endosomes and autophagosomes [43][44][45].Thus, it is possible that HOOK1 loss blocks autophagic flux by interfering with the proper func�oning of the HOPS complex.Studying the interac�on of HOOK1 with the HOPS complex in the future could help elucidate why autophagic flux might be par�ally impeded in our model.
Both excessive ac�va�on and blockade of autophagy can lead to cell death through apopto�c signaling in tumor cells [46,47].However, the ability of autophagy to suppress necro�c death is considered one of its most important prosurvival mechanisms [48,49], although other studies have shown that autophagy can promote necroptosis [50,51].Therefore, the involvement of autophagy in cell survival or death is complex and highly dependent on the cellular context.Generally, an increase in ER stress induces a protec�ve increase in autophagic flux to reduce proteotoxic stress and avoid cell death.Our results show that blocking autophagic flux affect cells with reduced HOOK1 levels to a greater extent than parental cells, leading to a significant increase in cell death, especially necrosis.This finding correlates with the role of autophagy as a suppressor of necro�c death and suggests that the autophagic pathway may act as a protec�ve mechanism against ER stress in our model.However, this prosurvival mechanism might not func�on perfectly, as the decrease in HOOK1 also appears to cause a par�al blockade of autophagic flux.Therefore, it may not be as effec�ve as expected in preven�ng cell death.
For instance, it could be due to altera�ons in autophagic flux.Both excessive ac�va�on and blockade of autophagy can lead to cell death through apopto�c signaling in tumor cells 36,37 .However, the ability of autophagy to suppress necro�c death is considered one of its most important prosurvival mechanisms 38,39 , although other studies have shown that autophagy can promote necroptosis 40,41 .Therefore, the involvement of autophagy in cell survival or death is complex and highly dependent on the cellular context.Generally, an increase in ER stress induces a protec�ve increase in autophagic flux to reduce proteotoxic stress and avoid cell death.To verify whether autophagy plays this protec�ve role when HOOK1 is reduced, we used chloroquine to completely block autophagic flux.We observed that the use of this drug affected cells with reduced HOOK1 levels to a greater extent than parental cells, leading to a significant increase in cell death (Figure 6B).Addi�onally, when autophagic flux is blocked, the preferred mode of cell death is necrosis.This finding correlates with the role of autophagy as a suppressor of necro�c death.These data suggest that the autophagic pathway may act as a protec�ve mechanism against ER stress in our model.However, the previous data indicate that this prosurvival mechanism may not func�on perfectly, as the decrease in HOOK1 also appears to cause a par�al blockade of autophagic flux.Therefore, it may not be as effec�ve as expected in preven�ng cell death (Figure 8).
The occurrence of ER stress and the problems in autophagic flux could also explain the changes observed in terms of CSC proper�es when HOOK1 is reduced.We observed that the downregula�on of this gene led to an increase in the death of EpCAM+-posi�ve cells in the ovarian cancer two cell lines studied (Figure 7A).This result suggests that the reduc�on in HOOK1, not only causes a decrease in the survival of ovarian cancer cells in general, but also specifically affects CSCs.Numerous studies have shown that maintaining ER proteostasis and autophagy flux intact is crucial for maintaining CSC integrity.For example, UPR induc�on can decrease CSC proper�es in colorectal carcinoma 42,43 , breast cancer 44 , head and neck cancer 45 , glioblastoma 46,47 , and prostate carcinoma 48 .Moreover, autophagy has been linked to the maintenance of CSCs in different tumors, such as breast cancer 49,50 , pancrea�c and liver carcinoma 51 , osteosarcoma 52 , ovarian cancer 53 , head and neck cancer 54 , and glioblastoma 55 .We observed in our model that increased ER stress or blockade of autophagy can reproduce the cell death of CSCs observed when HOOK1 is downregulated (Figure 7B).This finding further supports the hypothesis that the decrease in stem cell proper�es when HOOK1 is reduced may be due to this phenomenon these two phenomena.The par�al blockade of autophagy could also impact the survival of CSCs.Autophagy has been linked to the maintenance of CSCs in different tumors, such as breast cancer [59,60], pancrea�c and liver carcinoma [61], osteosarcoma [62], ovarian cancer [63], head and neck cancer [64], and glioblastoma [65].Consistent with these studies, we have observed that blocking autophagic flux with chloroquine also increases CSC death and reduces their propor�on within the tumor cell culture.
Given that reducing HOOK1 levels leads to the occurrence of proteotoxic stress and compromises the survival of CSCs, inhibi�ng this protein could be a promising therapeu�c strategy in ovarian tumors.In fact, we observed that the decrease in HOOK1 sensi�zes cells to compounds inducing ER stress and to proteasome inhibitors and that inhibi�ng HOOK1 prevented tumor forma�on in mouse models (Figure 4F).Drugs capable of inducing ER stress or blocking autophagy have been widely used in studies to treat different types of tumors.For ER stress, preclinical studies suggest the u�lity of potent ac�va�on of this mechanism to kill tumor cells 56-59 .In the case of autophagy, numerous studies have described that blocking this pathway can produce a cytotoxic effect in tumor cells.Two drugs that block the autophagic flux have been approved for clinical use, chloroquine and its deriva�ve hydroxychloroquine (HCQ) , and they have been used in clinical trials of adenocarcinoma, melanoma, colorectal carcinoma, myeloma, lymphoma, and renal carcinoma 60 .Hence, autophagy blockade is currently considered a promising poten�al strategy in the treatment of tumors refractory to conven�onal therapies.Based on the described use of drugs affec�ng proteostasis as therapeu�c strategies in cancer, HOOK1 could be a promising therapeu�c target, since reducing this protein affects mul�ple cellular processes capable of triggering proteotoxic stress in tumor cells.Moreover, we observed that inhibi�ng HOOK1 prevented tumor forma�on in mouse models (Figure 1E).Therefore, it would be interes�ng to study HOOK1 inhibi�on as a poten�al treatment for ovarian cancer, either as monotherapy or in combina�on with chemotherapeu�c agents or other drugs inducing proteotoxic stress, such as inhibitors of the proteasome or enhancers promoters of the UPR response.
(pages 14-18, lines 285-382) *References must be en�rely revised and reformated.Reffs #1 to #3 appear to be unrelated to the text, as a consequence the whole indexing is shi�ed and it is not possible to check the appropriateness of all the other ones.
We have reviewed the references to ensure that all of them have been correctly inserted into the text.
legend has been modified in order to give more insight into the different types of clones we observe and in what conditions the experiment was performed.New Fig legend reads as:

Figure 3 .
Figure 3. Hook1 downregulation causes a decrease in CSC-associated properties independent of the expression of genes traditionally linked to the CSC phenotype.(A) Analysis of the type of clones originated from a clonability assay of ovarian cancer cell lines with downregulated Hook1.The percentage of holoclones, meroclones and paraclones was assessed.Representative images of each type of clone are shown.(B) Analysis of the number and size of tumorspheres of ovarian cancer cells with downregulated Hook1.(C) Relative mRNA levels and (D) relative protein levels of factors traditionally associated with stemness in ovarian cancer cell lines with downregulated Hook1.The mean of 3 independent experiments ± SEM is represented.Statistical analysis was

Figure 7 .
Figure 7. Cell death induced by Hook1 downregulation may selectively impact CSCs.(A) Quantification by flow cytometry of the relative number of cells expressing the CSC marker EpCAM, EpCAM+ dead cells, and total dead cells in Hook1-downregulated cells.(B) Quantification by flow cytometry of the relative number of parental cells expressing the CSC marker EpCAM, EpCAM+ dead cells, and total dead cells after the treatment with an ER stress inducer (Brefeldin A) or an autophagic flux blocker (chloroquine).The mean of 3 independent experiments ± SEM is represented.Statistical analysis was performed with Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001).The absence of an asterisk means that the data are not statistically significant.

FIGURE 3 Figure 3 .
FIGURE 3Figure 3. Hook1 downregulation causes a decrease in CSC-associated properties independent of the expression of genes traditionally linked to the CSC phenotype.(A) Analysis of the type of clones originated from a clonability assay of ovarian cancer cells with downregulated Hook1.The percentage of holoclones, meroclones and paraclones was assessed.Representative images of each type of clone are shown.(B) Analysis of the number and size of tumorspheres of ovarian cancer cells with downregulated Hook1.(C) Relative mRNA levels and (D) relative protein levels of factors traditionally associated with stemness in ovarian cancer cell lines with downregulatedHook1.The mean of 3 independent experiments ± SEM is represented.Statistical analysis was performed with Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001).The absence of an asterisk means that the data are not statistically significant.
-8 and SKOV-3 cell lines from The Human Protein Atlas.(B) Quantification by flow cytometry of the percentage of cells expressing a selection of the CSC markers suggested for ovarian cancer in OVCAR-8 and SKOV-3 cells with HOOK1 downregulation.(C) Quantification by flow cytometry of the percentage of CD105+ cells in OVCAR-8 cell line and CD117+ cells in SKOV-3 cell line with downregulated HOOK1.

(
Fig. 1A: the labels are missing regarding the two groups in the boxplot The labels of Figure 1A have been included.

Fig
Fig 1B: Typos "Estrctural" The typo has been corrected.Fig 1B has been swapped with Fig 1C.Therefore, old Fig 1B is now new Fig 1C.

Figure 8 .
Figure 8. Interaction of HOOK1 with ER stress, autophagy, and cell death.Decreased HOOK1 levels lead to the activation of ER stress, which activates signaling pathways that result in increased cell death and autophagy.Autophagy acts as a protective mechanism for the cell, reducing protein load and thus contributing to cell survival.However, the decrease in HOOK1 may also interfere with autophagic flux, causing a partial blockage of it and, therefore, reducing the effectiveness of this mechanism in preventing cell death.The increased cell death affects both differentiated cells and CSCs, causing a reduction of the cell proliferation and CSC properties of ovarian cancer cells.
Figure 1.Hook1 is overexpressed in ovarian cancer patients, and its downregulation impairs the growth of ovarian cancer cells and abolishes the generation of tumors in vivo.(A) Comparison of the expression of HOOK11 in ovarian tumors vs. normal tissue.(B) Overall survival of patients with high vs. low expression of HOOK1.(C) Analysis of Hook1 alterations in various human tumors.(D) Validation of HOOK1 downregulation in ovarian cancer cells by WB and RT-qPCR analyses.(E) Colony formation ability and (F) growth curve analysis of ovarian cancer cells with Hook1 downregulation.The mean of 3 independent experiments ± SEM is presented.(G) Evaluation of tumor growth of xenografts generated fromHook1-downregulated cells in comparison to parental cell lines.Graphs represent the tumor size (mean ± SEM).Representative images of the tumors are shown.Statistical analysis was performed with Student's t test (*p < 0.05; **p < 0.01; ***p < 0.001).

MANUSCRIPT 5 .
Cell death induced by Hook1 downregula�on is dependent on the ER stress response and autophagic flux.

We propose a mechanism in which the maintenance of protein homeostasis by HOOK1 levels promotes the survival of ovarian cancer cells by regulating ER stress and autophagy.
We believe that HOOK1 is necessary to control cellular stress, and such stress can induce cell death, especially in those cells that are more sensitive, such as CSCs.

Supplementary Figure 2. Analysis of cell death, ER stress and autophagy in different HOOK1 CRISPR clones in OVCAR-8 and SKOV-3 cell lines. (A) Quantification
6(OVCAR-8) or 5x10 6 (SKOV-3) cells into the right flanks of four 4-week-old female athymic nude mice.Cells were suspended in Matrigel (Corning) prior to the injection.Animals were examined weekly.After 80-120 days, depending on the cell lines, mice were sacrificed, and tumors were extracted and conserved at -80 °C.Tumor volume (mm 3 ) was measured using calipers.All animal experiments were performed according to the experimental protocol approved by the IBIS and HUVR Institutional Animal Care and Use Committee (0309-N-15).Page 6: p-H2AX is more a direct marker of DNA double strand breaks and DNA replica�on stress than "classical" apoptosis, was there a reason to presume that HOOK1-downregula�on is inducing repair defects for DSBs?
Currently, no established link exists between the reduction in HOOK1 and defects in DSB repair mechanisms.We used p-H2AX as a marker for apoptosis because it plays a crucial role in DNA fragmentation during apoptosis and is phosphorylated at Ser139 by various kinases in response to apoptotic signals(Ref).Nevertheless, it is indeed noteworthy that pH2AX is an indicator of double-strand DNA breaks, suggesting potential future investigation into any closer association between decreased HOOK1 levels and replicative stress.Ref:• Mukherjee B, Kessinger C, Kobayashi J, Chen BP, Chen DJ, Chatterjee A, et al.DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells.DNA Repair (Amst).2006;5(5):575-90.• Solier S, Sordet O, Kohn KW, Pommier Y. Death receptor-induced activation of the Chk2and histone H2AX-associated DNA damage response pathways.Mol Cell Biol.2009;29(1):68-82.• Lu C, Zhu F, Cho YY, Tang F, Zykova T, Ma WY, et al. Cell apoptosis: requirement of H2AX in DNA ladder formation, but not for the activation of caspase-3.Mol Cell.2006;23(1):121-32.• Lu C, Shi Y, Wang Z, Song Z, Zhu M, Cai Q, et al.Serum starvation induces H2AX phosphorylation to regulate apoptosis via p38 MAPK pathway.FEBS Lett.