HSP70-2 is overexpressed in clinical samples of breast tumors
The expression of HSP70-2 mRNA and protein was examined in breast clinical cancer specimens by qRT-PCR and IHC respectively. HSP70-2 gene expression was detected in 83 % (128/154) of total breast cancer tissue specimens irrespective of clinicopathological features of breast cancer tissue specimens including histotypes, stages and grades but not in ANCT samples (Fig. 1a, Table 1). Congruent with RT-PCR data HSP70-2 protein expression was also detected in 83 % (128/154) tissue specimens (Fig. 1b) but not in matched ANCT (Additional file 2: Figure S1a, c). Notably, HSP70-2 expression was observed in 100 % of (8/8) DCIS, 83.4 % (116/139) of IDC, 80 % (4/5) of ILC and 100 % (2/2) of DCIS + IDC specimens. Furthermore, HSP70-2 expression was found in 100 % (3/3) of stage I, 80 % (68/85) of stage II, 86.7 % (39/45) of stage III and 100 % (6/6) stage IV of IDC histotypes of tissue specimens. HSP70-2 expression was detected in 89.8 % (62/69) of grade 1, 75 % (39/52) of grade 2 and 83.3 % (15/18) of grade 3 IDC specimens (Table 1). In addition, 80.4 % (41/51) of IDC specimens were found positive for HSP70-2 expression that had lymph node involvement (stage III and IV), whereas, 86.4 % (76/88) specimens with negative lymph node involvement (stage I and II) showed HSP70-2 expression (Table 1).
Based on immuno-reactivity score (IRS), the IDC specimens were divided in two groups as shown in Additional file 2: Figure S1d. Group I included specimens with >50 % cells expressing HSP70-2 protein, whereas, Group II included specimens with <50 % cells expressing this protein. Interestingly, number of patients (75.9 %, 88/116) expressing HSP70-2 was significantly higher (P < 0.0001) in Group I (IRS = 72.74 ± 1.34) compared to Group II patients (24.1 %, 28/116, IRS = 28.07 ± 1.89, Additional file 2: Figure S1d) irrespective of stages, grades and histotypes. Based on the HSP70-2 IRS score, after the Bonferroni correction for multiple comparisons, HSP70-2 protein expression were found to be significantly associated with grade 1 (63.71 ± 2.61) and grade 2 cases (63.74 ± 3.69, P < 0.029; Table 1). In addition, HSP70-2 protein expressing cells in patients with positive lymph node involvement (55.33 ± 3.44) and negative lymph node involvement (65.64 ± 2.56) was also observed.
HSP70-2 is over-expressed in breast cancer cell lines
Since breast tumor samples showed overexpression of HSP70-2 mRNA as well as protein, we next examined its expression in four different breast cancer cell lines viz., BT-474, MCF7, MDA-MB-231 and SK-BR-3. As shown in Fig. 1c, HSP70-2 mRNA expression was detected in all four breast cancer cells irrespective of their molecular phenotype but not in human normal mammary epithelial cells (HNMEC; Fig. 1c). There was higher HSP70-2 mRNA expression in triple negative MDA-MB-231 (>7 fold; P < 0.001) cells, followed by MCF7 (>3fold; P < 0.001), SK-BR-3 (>3 fold; P < 0.001) and BT-474 (1.28 fold; P < 0.05) with respect to HNMEC (Fig. 1d). Further Western blot analysis confirmed the expression of HSP70-2 protein in in all cell lines (Fig. 1e) along with their surface localization (Fig. 1f). In addition, we also observed cytoplasmic presence of HSP70-2 in all these cells (Additional file 2: Figure S1e) with specific localization in endoplasmic reticulum, mitochondria and Golgi bodies but not with nuclear envelope (Fig. 1g).
Down-regulation of HSP70-2 leads to reduced cellular proliferation, cell viability, colony forming ability of breast cancer cells
Interestingly we observed the overexpression of HSP70-2 in breast tumor as well as in breast cancer cell lines, therefore we next investigated its physiological relevance in oncogenic properties of cancer cells. Knock-down HSP70-2 expression was examined employing four HSP70-2 shRNA targets and scrambled shRNA (NC shRNA) in MCF7 and MDA-MB-231 cancer cells. Our qPCR and Western blot analysis clearly showed that HSP70-2 gene and protein expression was specifically down-regulated with HSP70-2 shRNA3 (P < 0.001; P < 0.002) and shRNA4 (P < 0.0001; P < 0.005) compared to HSP70-2 shRNA1, shRNA2 and NC shRNA (Fig. 2a) in MCF7 and MDA-MB-231 cells respectively. Therefore, in all subsequent in-vitro assays, HSP70-2 shRNA3 and shRNA4 targets were used in cell culture. Our cell proliferation studies revealed a significant reduction in cell count by 46.7 % (P < 0.0001) and 41.5 % (P < 0.0001) respectively in HSP70-2 shRNA3 and shRNA4 transfected MCF7 cells post 72h respectively as compared to NC shRNA transfected cells (Fig. 2b). Expectedly, MDA-MB-231 cells also showed reduction in cell count by 48.4 % (P < 0.05) in HSP70-2 shRNA3 transfected cells and 43.9 % (P < 0.001) in HSP70-2 shRNA4 transfected cells. Also, there was a significant reduction (P < 0.001) in cell viability of HSP70-2 shRNA3 and shRNA4 transfected MCF7 and MDA-MB-231 cells compared to NC shRNA transfected cells (Additional file 3: Figure S2a). Importantly, HSP70-2 depletion also resulted in a significant reduction in the colony forming ability of MCF7cells by 60.79 % (P < 0.0001) with HSP70-2 shRNA3 and 63.16 % (P < 0.0001) with HSP70-2 shRNA4 transfection (Additional file 3: Figure S2b). MDA-MB-231 cells also exhibited marked reduction of 60.01 % (P < 0.0001) and 63.87 % (P < 0.0001) in cells transfected with HSP70-2 shRNA3 and shRNA4 respectively (Additional file 3: Figure S2b). Thus, HSP70-2 seems to play an important role in cell proliferation, cell viability and tumorigensis.
Knockdown of HSP70-2 results in cell cycle arrest and induces senescence
Next we investigated the role of HSP70-2 in cell cycle. We observed that down-regulation of HSP70-2 expression led to accumulation of MDA-MB-231 cells in G0/G1 phase in HSP70-2 shRNA3 and shRNA4 treated cells (Fig. 2c and Additional file 3: Figure S2c). Further, phase contrast microscopy of these cells showed enhanced β-galactosidase staining in HSP70-2 depleted MDA-MB-231 cells (green color; Fig. 2d), while scanning electron microscopy (SEM) images showed flattened phenotype of these cells compared to normal (Fig. 2d ). Besides, HSP70-2 depleted MDA-MB-231 cells showed increased onset of senescence (P < 0.001; Fig. 2d) as also evident from enhanced expression of senescence associated marker, Decoy receptor 2 (DCR2) and lamin B1 in these cells (Fig. 2e). Thus, these results suggested that depletion of HSP70-2 seems to initiate senescence process.
We further examined the status of molecules involved in cell cycle arrest. Our qPCR results in HSP70-2 depleted MDA-MB-231 cells revealed 1.6 fold up-regulation of p21 expression (P < 0.019) with a concomitant reduction in CDK1 (P < 0.007), CDK2 (P < 0.009), CDK4 (P < 0.0003), CDK6 (P < 0.006), cyclin B1 (P < 0.027), cyclin D1 (P < 0.007) and cyclin E levels (P < 0.008; Additional file 3: Figure S2d). Further, Western blot analysis confirmed the over-expression of CDK inhibitors, p21 and decreased expression of cyclin D1, cyclin E, CDK4 and CDK6 and reduced expression of G2/M-phase CDK1, CDK2 and cyclin B1 and cyclin A2 in HSP70-2 depleted cells (Fig. 2e). We also observed decreased levels of phosphorylated Rb and corresponding accumulation of Rb due to G0/G1 arrest of the cells. As expected, proliferating cell nuclear antigen (PCNA) expression also decreased as shown in Fig. 2e. Collectively, these results suggested that G0/G1 arrest caused by ablation of HSP70-2 may be mediated through up-regulation of p21 and down-regulation of cyclins and their cognate kinases.
HSP70-2 gene silencing initiates apoptosis
The phenotypic changes associated with apoptosis were investigated by SEM in HSP70-2 depleted MCF7 and MDA-MB-231 cells. Post 24 h, both breast cancer cells exhibited early signs of apoptosis including cell shrinkage and blebbing, followed by the formation of apoptotic bodies in HSP70-2 shRNA3 and shRNA4 transfected cells (Fig. 3a). Post 48 h, most of the cells appeared to undergo apoptotic death. However, no phenotypic changes in the morphology of NC shRNA transfected (Fig. 3a) and lipofectamine treated cells were observed (Additional file 4: Figure S3a).
To elucidate the role of HSP70-2 in apoptosis, we analyzed several characteristic markers of apoptosis in HSP70-2 depleted cells. We analyzed annexinV staining to examine the externalization of phosphatidyl serine, an early sign of apoptosis. Our flow cytometry data showed marked increase in annexinV staining in MDA-MB-231 cells transfected with HSP70-2 shRNA3 and shRNA4 as compared to NC shRNA (Fig. 3b). We also determined changes in the mitochondrial membrane potential (MMP) of HSP70-2 depleted MDA-MB-231 cells using TMRE (tetramethyl rhodamine, ethyl ester) dye. Our data revealed loss of potential due to HSP70-2 shRNA treatment (Fig. 3c). Next, we investigated caspase cleavage by employing M30 assay and found higher population of M30 positive (Fig. 3d). To monitor the late apoptosis events, we also examined chromatin condensation by DAPI staining and observed a rise in the DAPI intensity in HSP70-2 depleted MDA-MB-231 cells (Fig. 3e). In addition, we examined DNA fragmentation by TUNEL assay and observed a marked increase in BrdU positive MDA-MB-231cells (Fig. 3f).
Analysis of pro-apoptotic gene expression by qPCR in HSP70-2 shRNA4 treated cells revealed a significant increase in the expression of BID (P < 0.003), caspase 6 (P < 0.033), caspase 7 (P < 0.008), caspase 9 (P < 0.012), PUMA (P < 0.002) and cytochrome-C (P < 0.007) by 1.39, 1.83, 1.72, 1.89, 3.0 and 1.92 folds respectively. Whereas the expression of anti-apoptotic molecules including BCL2 (P < 0.003), BCL-x
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(P < 0.045), Survivin (P < 0.009), cIAP2 (P < 0.003), XIAP (P < 0.035) and MCL1 (P < 0.007) was significantly inhibited (Additional file 4: Figure S3b). Further, Western blot analysis revealed increased expression of molecules involved in intrinsic pathway including caspase 3, caspase 6, caspase 7, caspase 9 and cytochrome-C (Fig. 3g). The levels of pro-apoptotic molecules including BID, BAD, BAK, BAX, NOXA, APAF1 and PUMA were also up-regulated in these cells. As expected, low level of expression of several anti-apoptotic molecules including BCL2, BCL-xL, Survivin, MCL1 and XIAP was observed (Fig. 3g). Interestingly, increased expression of AIF and PARP-1 in these cells suggested the activation of caspase-independent pathway under these conditions (Fig. 3g). Considering the important role of HSP70-2 in protein folding and degradation, activation of intrinsic apoptotic pathway due to HSP70-2 abrogation prompted us to investigate whether the ER stress could be the underlying cause of pro-apoptotic cell death. We did find decrease protein expression of ER chaperone protein, GRP78 in HSP70-2 depleted cells (Fig. 3g) suggesting its essential role in cell survival.
HSP70-2 is essential for cellular motility, migration and invasion
Increased cellular motility, migration and invasion are distinguishing features of cancer cells. We studied transwell membrane assays to study the effect of ablation of HSP70-2 on migratory and invasive properties of MCF7 and MDA-MB-231cells. The cell migration assay exhibited a significant reduction (P < 0.05) in the migration of HSP70-2 shRNA3 and shRNA4 transfected cells compared to NC shRNA (Fig. 4a, b) with a concomitant loss of invasive ability through matrigel (P < 0.05, Fig. 4c, d). Further, the SEM images of transwell membranes confirmed reduced migration of these cells (Fig. 4e, f). In addition, wound healing assay also indicated reduced cellular motility under the conditions as compare to control cells (Additional file 5: Figure S4a).
Epithelial-Mesenchymal Transition (EMT) is considered to be a benchmark in cancerous growth. Therefore, we measured the mRNA expression of EMT markers in HSP70-2 depleted cells. As shown in Additional file 5: Figure S4b, there was an overall significant reduction in the mRNA levels of mesenchymal markers such as N-Cadherin (P < 0.0001), P-Cadherin (P < 0.012), MMP2 (P < 0.0001), MMP3 (P < 0.004), SLUG (P < 0.006), SNAIL (P < 0.049), Vimentin (P < 0.005) and SMA (P < 0.010). However the epithelial cell marker, E-Cadherin (P < 0.002), showed an increased expression of 2.74 fold (Additional file 5: Figure S4b). Further, Western blot analysis validated our qPCR data revealing down-regulation of SNAIL, SLUG and TWIST (EMT regulators) along with SMA, Vimentin, N-Cadherin, P-Cadherin, MMP2, MMP3 and MMP9 (Fig. 4g). As expected, there was increase in E-Cadherin expression (Fig. 4g). Thus, HSP70-2 seems to play an important role in cellular migration and invasion orchestrated via EMT pathway.
Depletion of HSP70-2 causes reduced xenograft breast tumor growth in-vivo
To validate our finding on HSP70-2 in cell culture, we studied the effect of ablation of HSP70-2 on tumor growth in xenograft mouse model. As shown in Fig. 5a, there was a reduction in tumor size in HSP70-2 shRNA4 treated mice compared to NC shRNA treated mice. The tumor weight and volume of mice injected with HSP70-2 shRNA4 was significantly reduced (P < 0.001) as compared control animals (Fig. 5b, c). Western blot analysis of xenograft tissue sections showed that HSP70-2 protein and PCNA was reduced post HSP70-2 shRNA4 administration (Fig. 5d). Further, IHC analysis of excised tumor sections confirmed reduction in HSP70-2 and PCNA expression (Fig. 5e).
We further examined these tumor sections to understand changes in the molecules involved in various pathways following HSP70-2 depletion by IHC. In agreement with our in-vitro data, the IHC analysis showed increased expression of CDK inhibitor, p21 in mice administered with HSP70-2 shRNA4 compared to NC shRNA treated mice (Fig. 5e). This was accompanied with decreased expression of CDKs including CDK1, CDK2, CDK4 and CDK6, cyclins including cyclin A2, cyclin B1, cyclin D1 and cyclin E in tumors treated with HSP70-2 shRNA4 in contrast to NC shRNA. Next, we compared the expression of molecules involved in apoptosis in HSP70-2 shRNA4 and NC shRNA treated tumor (Fig. 6). Notably, most of the key molecules of apoptotic pathway showed increased expression of caspase 3, caspase 6, caspase 7, caspase 9, cytochrome-C, APAF1, BAD, BAX, BID, PUMA and NOXA in HSP70-2 shRNA treated tumors (Fig. 6). This was also concomitant with down-regulation of several anti-apoptotic molecules, BCL2, BCL-xL, MCL1, Survivin, cIAP2 and XIAP (Fig. 6). In addition, caspase independent AIF mediated cell death also increased as revealed by increased expression of AIF and PARP1 in HSP70-2 shRNA treated compared to NC shRNA treated tumors (Fig. 6). Also we found, reduction in expression of glucose-regulated protein, GRP78 in tumors treated with HSP70-2 shRNA4 (Fig. 6).
We further investigated the expression of EMT regulators. We observed reduction in the expression of EMT regulator SNAIL, in consequence to the ablation of HSP70-2. In addition, there was a decreased expression of mesenchymal markers such as N-Cadherin, P-Cadherin, Vimentin, SNAIL, SLUG, TWIST and SMA in HSP70-2 shRNA4 treated tumor compared to NC shRNA treated tumors. HSP70-2 ablation also revealed increase in epithelial marker, E-Cadherin expression (Fig. 6). Our data revealed that HSP70-2 shRNA4 treated tumors exhibited decreased expression of MMP2, MMP3 and MMP9 (Fig. 6). To validate our IHC finding, we employed qPCR to check mRNA expression of several key molecules in HSP70-2 shRNA4 treated tumors, relative to the NC shRNA treated tumors. It is interesting to note that our qPCR results were consistent with IHC analysis. Initially we checked the relative level of mRNA of HSP70-2 in HSP70-2 shRNA4 treated tumors and NC shRNA treated tumors and found a marked depletion of HSP70-2 mRNA expression upon shRNA mediated gene silencing (Additional file 6: Figure S5a). There was a reduction in CDK1 (P < 0.028), CDK2 (P < 0.039), CDK, 4 (P < 0.001), CDK6 (P < 0.016), cyclin B1 (P < 0.001), cyclin D1 (P < 0.001) and cyclin E (P < 0.001) expression, relative to the NC shRNA treated tumors (Additional file 6: Figure S5b). Our qPCR study on apoptotic molecules in HSP70-2 shRNA4 treated tumors revealed an increase expression of BAD (P < 0.004), caspase 6 (P < 0.029), caspase 7 (P < 0.007), caspase 9 (P < 0.016), PUMA (P < 0.008), NOXA (P < 0.001) and cytochrome-C (P < 0.001). Also decreased expression of anti-apoptotic molecules including BCL2 (P < 0.001), BCL-x
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(P < 0.001), Survivin (P < 0.028), MCL1 (P < 0.005) and cIAP2 (P < 0.001) were found (Additional file 6: Figure S5c). Comparison of mRNA expression of EMT molecules in HSP70-2 shRNA treated tumors and NC shRNA treated tumors revealed decreased expression of SLUG (P < 0.013), P-Cadherin (P < 0.038), N-Cadherin (P < 0.001), MMP3 (P < 0.001), MMP9 (P < 0.001), SMA (P < 0.029) and SNAIL (P < 0.008). However, E-Cadherin (P < 0.001), displayed increased expression of 2.55 fold in HSP70-2 shRNA treated mice (Additional file 6: Figure S5d). Interestingly, our IHC data and qPCR results showed similar results.
Collectively, our results indicated that HSP70-2 contributes in cellular proliferation, cellular migration and invasion and its depletion causes significant reduction in these processes both in-vitro and in-vivo xenograft mouse model.