Transcription Factor SOX2Predicts Prognosis And Regulates Cancer Phenotypes Via A PTEN-RB Pathway In Gastric Carcinoma

During the ensuing decade transcription factor SOX2is solidified as one of the hallmarkparticipants throughout the developmental process in stomach. Ectopic SOX2levels areresponsible for exerting confounding impacts that enable normal cells to becometumorigenic and ultimately malignant on multistep evolution of human gastric carcinoma(GC). We thus identify SOX2expression profiling over the course of a GC lifespan,encompassing the contributions of SOX2to our understanding of GC tumorigenesis andprognosis. In addition, the essence of transcription factor regulation exhibited by SOX2has served both to clarify and modulate the original formulation of cancer phenotypes in GC.Here a central role for SOX2that governs GC establishment and progression reflected onchallenges arising in analogous studies and highlighted mechanistic concepts that might beintegral to a more rational elaboration of SOX2-associated traits in GC.【Aims】To identify the expression levels of SOX2protein inhuman specimens throughoutmilestone stages of gastric carcinoma progression, and to uncover the specific role oftranscription factor SOX2in GC tumorigenesis and progression, for a greaterunderstandingof the underlying mechanisms of SOX2in GC and a more substantial leap for taking SOX2as a novel predictor in GC prognosis druing clinical practice in near future.【Methods】1. To determine SOX2that might participate in GC progression rather than passivebystanders, the heterogeneity of SOX2levels was detected by western blot andimmunohistochemistry in human gastric specimens stratified by pathological status.2. Given the correlations between SOX2expression and clinical progression, we assessedthe prognostic roles for SOX2elaborately for further characterization byimmunohistochemistry, western blot and qRT-PCR.3. To better enumerate SOX2-relevant features, we stably expressed SOX2in MKN28human gastric cancer cells. This over expression ensured SOX2levels comparable tothose in MKN28cells carrying control vectors. By means of WST-1, we checked theeffects of elevated SOX2on GC cell proliferation in vitro. Apart from in vitro properties,MKN28cells expressing SOX2and control cells were injected into intravenous sites ofthe nude mice, partially for a direct recording of primary tumor growth, and partially forKi67staining on primary tumors tissues.4. Cell cycle and apoptosis analysis of MKN28cells with and without SOX2overexpression were conducted to discern any reactive attenuation in vitro, while in vivoapoptotic potential was examined by TUNEL staining on SOX2-expressing tissuespecimens from primary mice tumors. 5. The invasive capability was testified by cell invasion and migration assays in vitro andtail vein injection in vivo respectively.6. To uncover the mechanistic underpinning of SOX2, we applied two high-throughputgenome-sequencing analyses---ChIP-DLS and cDNA expression microarray---inSOX2-expressing MKN28cells to reveal putative targets catering to the same criteriathat they not only straight attach to SOX2promoter but also manage to take on aremarkable expression alteration subsequent to SOX2over expression. Guided bybio-information analysis, we culminate in the acquisition of our favorable candidate.7. To verify the validity of our prediction, we deployed a series of approaches likeChIP-PCR, EMSA and luciferase report assays.8. Furthermore, we suppressed the expression of our predicted target with siRNA inSOX2-overexpressing MKN28cells to re-confirm our deduction that inhibition of thepredicted target might compensate a cohort of functional traits subject to SOX2overexpression, which was supposed to exemplify both in vitro and in vivo attributes of GCcell proliferation, apoptosis, cell invasion and migration.9. Finally the association of PTEN and p-RB levels with that of SOX2was assayed withimmunohistochemistry in primary human GC and tumor-matched adjacent gastrictissues to answer whether contributions of SOX2, PTEN and p-RB expressioncorrelations are well suited to be a relevant prognostic indicator group during the courseof GC progression.【Results】1. The preceding observations demonstrated that SOX2expressions were specificallydiminished with GC progression, which included normal gastric tissues and tissuesderived from chronic gastritis,chronic atrophic gastritis (CAG) with intestinalmetaplasia,CAG with mild dysplasia of epithelium, gastric adenocarcinoma andmetastatic adenocarcinoma from stomach. The association of low SOX2levels with GC persisted dependent of both tumor gradeand molecular subtype. Such grade and subtype dependence avails SOX2of beingclinically utilized as a prognostic marker for human GC.2. Furthermore, elevated SOX2did impede proliferation, invasiveness and metastasis invitro and in vivo. Also, SOX2over expression enhances apoptosis but did not affect cellcycle. These effects were exclusively attributable to the biological activities of SOX2, asindicated by the fact that equivalent overexpression of a control vector failed to imposeinfluences alike.3. We proved that SOX2’s ability to function in GC derived from its potency to up-regulatePTEN expression, which renders RB protein de-phosphorylated.4. The coordinate differential levels of these3functionally relevant contributors affordedthe most pronounced clinical implications to GC progression and overall survival. Ofnote, it is intensely suggested that within this cohort, the full spectrum of reflection onworse survival are elicited via a target signature based on not only a concomitantreduction of SOX2and PTEN levels but also a concordant identification of an increasedp-RB level in human GC.【Conclusions】1. Our compelling body of evidence highlighted that an ever-declining expression of SOX2acted at early stages of human gastric malignancies.2. The best-characterized alteration of SOX2profiling in GC can fully recapitulate clinicalmalignancy and outcome. When GC specimens were stratified based on clinical status,we read that for SOX2expression in primary tumor tissues of a GC patient wasinversely proportional to disease progression, when compared to the SOX2level inmatched adjacent gastric tissues of the same patient; moreover, metastatic GC patientsdisplaying low SOX2levels in their metastases normally ended up with even worseclinical prognosis such as diminished distant-free survival comparing to non-metastaticGC patients with low SOX2expression in their primary tumors.The convergence of our findings helps to discover a notion that a subsequentup-regulation of PTEN triggered by SOX2over expression in GC cells serves tohyperactive the de-phosphorylation of p-RB protein, by which heterotypic interactions 3. give rise to SOX2-imposed capacities of cell apoptosis promotion and concomitantsuppression over cell proliferation and metastasis in human GC.