Role of the SET/PP2A signaling axis and its microRNA-mediated deregulation in colorectal cancer development : Potential clinical an therapeutic impact

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SUMMARY (ABSTRACT) Introduction Colorectal cancer (CRC) is a common and lethal disease. One of the fundamental processes driving the initiation and progression of CRC is the accumulation of a variety of genetic and epigenetic changes in colonic epithelial cells. Over the past decade, major advances have been made in our understanding of cancer genetics and epigenetics, including microRNA (miRNA or miR) deregulation. As with gene mutations in the cancer genome, a subset of these epigenetic alterations, affecting miRs, are presumed to have a functional role in CRC. Those improvements in our knowledge of CRC pathogenesis have led to these alterations being developed as clinical biomarkers for prognostic and therapeutic applications. Progress in this field suggests that a better comprehension of CRC molecular landscape will be commonly used in the near future to direct the management of CRC. Despite this great progress, further molecular and clinical research is needed to better select patients for current and novel emerging therapies and also to more specifically design future clinical trials based on alternative molecular target therapies under investigation. Protein phosphatase 2A (PP2A) is a tumor suppressor consisting in a heterotrimeric complex that regulates many signaling pathways crucial for cancer cell transformation. Several distinct PP2A inhibitory mechanisms had been identified in tumor cells such as phosphorylation and/or deregulation of some PP2A subunits, miRs deregulation and overexpression of endogenous PP2A inhibitors. Those alterations had been reported in many cancer types as contributing mechanisms of malignancy and conferring a poor prognosis to some tumors. However, the importance and potential clinical value of those alterations as biomarkers for CRC as well as their role in the pathogenesis of metastatic CRC (mCRC) remained mostly unexplored. Moreover, miR-21 and miR-31 are miRs with an oncogenic function that had been reported as potential regulators of PP2A activity. Their deregulation has been described to play an important role in CRC development and they have been associated with 5- Fluorouracil (5-FU) resistance. However, the role of these miRs as predictive biomarkers of response to 5-FU based neoadjuvant chemoradiotherapy (CRT) in locally advanced rectal cancer (LARC) remained unexplored. In this PhD research, we investigated the role of PP2A and its regulatory mechanisms in CRC pathogenesis. Furthermore, we analyzed the potential impact of PP2A inhibition based biomarkers in mCRC and LARC. Material and methods In vitro studies: We used 9 types of human CRC cell lines and we performed several in vitro studies such as proliferation and cell viability assays, phosphatase activity assays, analysis of caspase activation, transfection experiments, colony-forming assays, western blot analysis and luciferase assays. Patient samples: To validate our in vitro results and to explore the potential clinical value of our proposed biomarkers, the study comprised fresh-frozen samples and formalin-fixed paraffin-embedded (FFPE) tumor samples of 522 CRC patients divided in 4 independent CRC patient cohorts that are explained in more detail later. To measure miR expression levels in tumor samples we performed real-time RT-PCR and SET, PP2A and p-PP2A was measured by both RT-PCR and immunohistochemistry (IHC). All the manuscripts elaboration was carried out in accordance with the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines. Results In the first paper, we show that PP2A is frequently inactivated in patients with CRC. Moreover, we identified overexpression of the endogenous PP2A inhibitors SET and CIP2A and downregulation of the regulatory PP2A subunits PPP2R2A and PPP2R5E, as contributing mechanisms to PP2A inhibition in CRC. Thus, we further studied the relevance of each of those PP2A inhibitory mechanisms in CRC. In the second paper, we reported that phosphorylated protein phosphatase 2A at tyrosine 307 (p-PP2A-C Y307) is a molecular mechanism that contributes to PP2A inhibition in CRC and confers a worse outcome. This alteration was found in 17.2% of mCRC and the subgroup of high p-PP2A levels patients showed substantially worse overall survival (OS) (median 0S, 6.0 vs. 26.2 months, P< 0.001) and progression-free survival (PFS) (median PFS, 3.8 vs. 13.3 P< 0.001). Importantly, the multivariable analysis demonstrated that p-PP2A is an unfavorable independent factor associated with OS (hazard ratio 2.7; 95% confidence interval, 1.8 – 4.1; P< 0.001) and PFS (hazard ratio 3.0; 95% confidence interval; 1.8-5.0; P<0.001). In the third paper, we also reported that the restoration of p-PP2A using PP2A activating drugs such as forskolin is a potential alternative therapy with benefit in patients with high p-PP2A. In the fourth paper, we showed that SET overexpression promotes cell growth and colonosphere formation. Furthermore, we published that SET inhibits PP2A then impairing its antitumor effects and SET reduces sensitivity to oxaliplatin in CRC cell lines. Moreover, SET overexpression was detected in 24.8% of mCRC patients, determined significantly shorter OS (8.6 vs. 27 months, P< 0.001), PFS (7.1 vs. 13.7 months; P< 0.001) and poor response to oxaliplatin-based chemotherapy. To continue, in the fifth paper we show that miR-199b is downregulated in 4 out of 5 SET overexpressing CRC cell lines and that miR-199b expression is inversely correlated with SET overexpression in CRC patients. Moreover, we published that miR-199b led to PP2A activation through a SET inhibition impairing cell viability and enhancing oxaliplatin sensitivity in CRC cells. MiR-199b was found downregulated in 25% of cases, and associated with lymph metastasis (P= 0.049), presence of synchronous metastasis at diagnosis (P= 0.026) and SET overexpression (P< 0.001). Furthermore, low miR-199b levels determined shorter OS (30 months vs. 9.7 months, P< 0.001), PFS (15.4 vs. 8.6, P= 0.003) and predicted clinical benefit to oxaliplatin treatment. Finally, in papers sixth and seventh we showed how the potential PP2A regulators, miR-21 and miR-31, when they are found overexpressed are able to predict pathological response and outcome to neoadjuvant 5-FU based CRT in LARC. MiR-21 was found overexpressed in 77.6 % of LARC cases, and significantly correlated with pathological response (P= 0.013). The odds ratio of having miR-21 overexpression and not getting a pathological respond to CRT resulted in 9.75 (confidence interval (CI) 2.24 to 42). Sensitivity, specificity, negative predictive values, and positive predictive value were 86.6, 60, 42.8, and 92%, respectively. Multivariate analysis confirmed the clinical significance of miR-21 determining preoperative CRT response. In the other hand, high miR-31 levels were found overexpressed in 34.2% of cases. Its overexpression significantly predicted poor pathological response (P= 0.018) and a worse OS since 78% of the patients with high miR-31 expression were alive at 6 years follow up compared with 96% of those with low miR-31 (P= 0.008). Conclusions Altogether we have identified, the tumor suppressor activity of PP2A is commonly inhibited in CRC. SET overexpression, p-PP2A-C Y307, miR-199b downregulation, and the downregulation of the PP2A regulatory subunits PPP2R5E and PPP2R2E as contributing mechanisms to this PP2A inhibition in CRC. In addition, high levels of p-PP2A-C Y307 is an alteration that determines poor outcome in mCRC and SET overexpression is a frequent event in mCRC that plays a potential oncogenic role associated with worse outcome and resistance to oxaliplatin. In the same way, miR-199b is a tumor suppressor miR whose downregulation independently determines worse outcome in mCRC and resistance to oxaliplatin therapy. Finally, deregulation of the PP2A potential modulators, miR-21 and miR-31, have clinical impact determining pathological response and outcome in LARC.