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  • Increasing evidence has strongly suggested that genetic vari

    2019-08-26

    Increasing evidence has strongly suggested that genetic variants in classical PTP gene family could play an important role in carcinogenesis. However, previous studies often explored effects of single or several classical PTPs, rare systematic analysis has investigated the association of classical PTP gene family with CRC risk. Hence, we hypothesized that missense variants in classical PTP gene family could modulate the susceptibility to CRC by influencing protein function. A bioinformatics analysis was initially applied to systematically search genetic missense variants in classical PTP genes, and then a two-stage case-control study was performed to examine the association of candidate variants with CRC risk in a Chinese population. Finally, we explored the potentially pathogenic mechanism of verified variants by functional assays.
    Subjects and methods
    Results
    Discussion In this study, we integrated bioinformatic analyses, exome-wide association data and biochemical assays to investigate the association of missense variants in the classical PTP gene family with CRC risk. Population study revealed that PTPN12 rs3750050 significantly modified the risk of CRC. Further biochemical assays indicated that rs3750050 G (T573 A) significantly impaired PTPN12’s dephosphorylation on Ras/MEK/ERK signaling, then accelerate G1/S transition via up-regulation of CCND1 expression, thus making effect on the pathogenesis of CRC. In stage one, we identified two promising variants. One was PTPN23 rs6780013, but it failed to pass the verification of stage two. This variant is located in a susceptible region 3p21.31 reported by a recent CRC GWAS [20]. Unfortunately, evidence from our study was insufficient to support the association of PTPN23 and CRC risk. Another was PTPN12 rs3750050, which was successfully validated in both stages and subsequent functional assays. Protein PTPN12 is a cytoplasmic tyrosine phosphatase expressed ubiquitously that efficiently dephosphorylate specific tyrosine substrates, thus playing a part in in regulating cell growth, proliferation and AZD8931 [[29], [30], [31], [32]]. In 2011, Sun first showed PTPN12 as a new tumor suppressor gene and showed that loss of this protein resulted in activation of multiple PTKs in breast cancer [19]. Recently, Kwiatkowski also reported that PTPN12 is a novel candidate gene for early-onset CRC susceptibility [33]. Our study not only supported the contributing role of PTPN12 in CRC susceptibility, but also further revealed how the functional variant impaired the tumor-suppressor effect of PTPN12 in CRC susceptibility. PTPN12 rs3750050 is located within the proline-rich region adjacent to C-terminal of this protein (573aa). Charest A reported that the motif (576˜613aa) of mouse PTPN12 is necessary for SHC-PTPN12 interaction [34]. Comparing nucleotide sequences of the human, mouse, rat, yak and Pteropus PTPN12 protein, we find the motif (576˜613aa) is high conservative. Moreover, Tyr573 which is located near the motif also show extreme evolutionary conservation. So, we speculated that although this variant does not occur in the catalytic domain (28-293aa), the variant might impair the regulation of SHC by PTPN12. Our experiments provided a concrete evidence line to illustrate a proposed oncogenic role of rs3750050 in CRC (Fig. 3). In normal condition, IGF1 stimulates the initiation of Ras/Raf/ERK signaling pathway through phosphorylating the adaptor protein p52 SHC. Then SHC recruits GRB2 to constitute complex adaptors, which relay and amplify an exquisitely fine-tuned regulation of multiple downstream effectors, including Ras, Raf, MEK and ERK. Activated ERK phosphorylates AP-1 to upregulate cell cycle regulators, and eventually accelerates G1 to S phase transition. At the end of G1 phase, low-phosphorylated Rb protein binds to E2F–HDAC complex to silent its transcriptional activity, inducing cell into checkpoint. CCND1 encodes cyclin D1, a crucial subunit of cyclin D1-CDK4/6 complex, which could phosphorylate Rb, dissociate the silent complex and then promote G1/S transition. Recent studies have revealed that cyclin D1 induction is a major way for Ras/MEK/ERK signaling to promote G1/S transition [35]. Thus we selected CCND1 as the testing effector. Faisal A and Habib T’ studies both suggest that PTPN12 negatively regulates Ras/Raf/ERK signaling cascade by dephosphorylating activated-SHC protein [36,37]. When a functional damage (e.g. rs3750050) occurs in PTPN12, the mutant PTPN12 fails to dephosphorylate activated-SHC, impairs its inhibitory regulation on Ras/Raf/ERK pathway, then increases the expression of CCND1, and ultimately leads to aberrant cell proliferation. Our findings might suggest tightly regulation of RTKs by PTPN12 might be critical, whose dysregulation contributes to colorectal carcinogenesis.