The role of mTOR and phospho-p70S6K in pathogenesis and progression of gastric carcinomas: an immunohistochemical study on tissue microarray

Gastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century. It continues to be a major health problem because of the slow decrease in incidence in Asia and high mortality of diagnosed gastric carcinoma in West [1]. Therefore, it is of much significance for the prevention, treatment and prognosis evaluation of gastric cancer to clarify its molecular mechanisms and find out a good biomarker to indicate its carcinogenesis and subsequent progression.

The mammalian target of rapamycin (mTOR) also known as FK506 binding protein 12-rapamycin associated protein 1 is a serine/threonine protein kinase that supports cell growth, cellular metabolism, cell proliferation, cell motility, cell survival, protein synthesis, and transcription such as angiogenesis and autophagy. mTOR that is an evolutionarily conserved serine-threonine kinase of a 289-kDa in length belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. mTOR is composed of an N-term; 20 tandem repeats-HEAT which are implicated in protein-protein interactions; and a C-term which includes a FAT domain, a FBR domain, a kinase domain, a NDR domain and a FATC domain. The FATC domain is essential to mTOR activity and the deletion of a single amino acid from this domain abrogates the activity. mTOR can be autophosphorylated via its intrinsic serine/threonine kinase activity. mTOR exerts its multiple functions in the context of two different multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR, Raptor, mLST8, and PRAS40, and importantly activates p70 ribosomal protein S6 kinase and inactivates eIF4E binding protein 1, which promotes protein translation and cell growth. Conversely, mTORC2 is composed of mTOR, Rictor, Sin1, and mLST8, phosphorylates and activates another member of the AGC kinase family, Akt. Current research indicates that mTOR integrates the input from multiple upstream pathways, including insulin, growth factors (such as IGF-1 and IGF-2), and mitogens. mTOR also functions as a sensor of cellular nutrient and energy levels and redox status [2–5].

P70 S6 kinase (p70S6K) is activated in a signaling pathway that includes mTOR. P70S6K is a mitogen-activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression. This kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains and subsequently phosphorylates specifically ribosomal protein S6. Activation occurs via phosphorylation at ser411, Thr421 and Ser424 within the pseudosubstrate region. Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function. Stimulation of mammalian cells by a variety of mitogenic stimuli results in a rapid, biphasic activation of p70S6K. Inhibition of p70 activity inhibits the entry into S phase of the cell cycle and exhibits cell cycle arrest at G0/G1 phase, suggesting that the activation of p70S6k plays an obligatory role in mediating mitogenic signals during cell activation [6–8].

mTOR signaling pathway and its downstream serine/threonine kinase p70S6k were frequently activated in human cancers and the dysregulation of the mTOR pathway is implicated as a contributing factor to various human disease processes, especially various types of cancer[5, 6, 8–11]. However, the mTOR/p70S6K signaling pathway in gastric carcinomas has not been investigated so far, which impel the authors to investigate the role of mTOR/p70S6K signaling pathway for finding a novel target for the anticancer drugs in gastric carcinoma. In the present study, we observed that mTOR and P70S6K expression were examined in gastric carcinoma, adjacent non-tumorous mucosaand adenoma, and compared with the clinicopathological parameters of tumors to explore the clinicopathological significance and molecular role of the mTOR signal pathway in the stepwise development of gastric carcinomas.

Mammalian target of rapamycin (mTOR) is also known as FKBP-rapamycin-associated protein or rapamycin and FKBP target and functions as a serine/threonine protein kinase to sense adenosine triphosphate and amino acids to balance nutrient availability and cell growth. When sufficient nutrients are available, mTOR is phosphorylated via the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, transmits a positive signal to p70 S6 kinase (p70S6K), and participates in the inactivation of the eukaryotic translation initiation factor 4E inhibitor, 4EBP1. Therefore, mTOR plays a key role in cellular growth and homeostasis, and its regulation is frequently altered in tumors [8, 15].

Although mTOR protein can shuttle between the nucleus and cytoplasm [16, 17], we only observed its cytoplasmic distribution in line with the figure of its antibody data sheet. The phenomenon might be due to cell specificity and different antibody. In the present study, the antibody was produced with a synthetic peptide corresponding to residues near the C-term of PI3K/PI4K domain of human mTOR/FRAP as an immunogen. In addition, we found no difference in mTOR expression between gastric ANTC, adenoma and carcinoma, which suggested its role of growth-regulating in all gastric epithelial and lesion cells. However, its active form, phosphorylated mTOR might contribute to the carcinogenesis according to the literature [18–23]. In contrast, its down-stream target, the aberrant expression of cytoplasmic and nuclear phoshorylated p70S6K occurred in gastric adenoma-adenocarcinoma sequence. For instance, adenoma displayed higher cytoplasmic expression of active p70S6K than NNM group, whereas nuclear counterpart was less expressed in carcinoma than NNM and adenoma. It was speculated that different subcelluar distribution of phospho-p70S6K might have distinct biological function in the malignant transformation of gastric epithelial cells.

The 70-kDa S6 kinase (p70S6K) is a cytoplasmic Ser/Thr kinase that is mainly known to regulate protein translation through phosphorylation of the 40S ribosomal protein S6. Activation of p70S6K is achieved through phosphorylation on multiple Ser/Thr residues by stimulation with growth factors such as epidermal growth factor (EGF), thrombin, and lysophosphatidic acid (LPA)[23, 24]. To the role of phopsho-p0S6K protein in the progression of gastric carcinoma, its expression was compared with the aggressive behaviors of carcinoma and for the first time found that nuclear phosphor-P70S6K expression was inversely linked to tumor size, depth of invasion, lymph node metastasis and UICC staging. It was suggested that down-regulated expression of nuclear phospho-P70S6K was involved in the growth, invasion and metastasis of gastric carcinoma and might be employed to indicate the biological behaviors of gastric carcinoma in clinicopathological practice.

Although gastric cancer is malignant tumor originating from the same gastric epithelium, its morphological features vary substantially with the individual patients [13]. According to Lauren's classification, intestinal-type carcinomas are characterized by cohesive carcinoma cells forming gland-like tubular structures with expanding or infiltrative growth pattern. The cell cohesion is less apparent or absent in diffuse-type carcinoma and cancer cells diffusely spread in the gastric wall lesions. Tumors that contain approximately equal quantities of intestinal and diffuse components are called mixed carcinoma [13, 14]. These three markers were preferably expressed in the older patients with gastric cancer and intestinal-type carcinoma. Here, it was noted that mTOR, cytoplasmic and nuclear P70SK6 expression was higher in intestinal-than diffuse-type carcinomas, indicating that these three markers might play an important role in intestinal-type carcinogenesis, but less in de novo carcinogenic pathway and underlie the molecular basis for differentiation of both carcinomas.

To clarify the prognostic significance of mTOR, cytoplasmic or nuclear P70S6K expression, we here analyzed their relation with the survival of 412 patients with gastric carcinoma and found a close relationship link between the positivity of mTOR and nuclear phospho-P70S6K expression and favorable survival. Multivariate analysis demonstrated six independent prognostic factors such as age, depth of invasion, lymphatic invasion, lymph node metastasis, Lauren's classification and mTOR expression were independent prognostic factors for overall gastric carcinomas. However, several evidences indicated that phosphor-mTOR expression was closely linked to the poor prognosis of the patients with cervical adenocarcioma or hepatocellular carcinoma [18, 25]. PS6K overexpression was reported to correlate with worse distant disease-free survival of early-stage breast cancer patients. The paradoxical phenomenon might be attributed to the different mTOR types or different subcellular distribution of p70S6K protein. Here, nuclear p70S6K was inversely related to the tumor size, depth of invasion, lymph node metastasis and UICC staging, which are aggressive appearances of gastric cancer. The finding indicated p70 S6 phosphorylation in the nucleus might play some inhibitory role in gastric cancer and subsequent progression distinct from that in the cytoplasm.

Aberrant expression of p-P70S6K might play an important role of malignant transformation of gastric epithelial cells and was closely related to growth, invasion, metastasis and prognosis of gastric carcinomas and was considered as a promising marker to indicate the pathobiological behaviors. The distinct expression of mTOR and p-P70S6K could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. Nuclear p-p70S6K was a good marker to indicate the favorable prognosis of gastric carcinoma patients, albeit dependent on other parameters, but mTOR expression was an independent factor for the prognosis.