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  • br of these CRC cells promoted by


    of these CRC ATPγS promoted by CPA4 was reversed after treatment with cryptotanshinone or U0126 (Fig. 6B–F). The above results may show that CPA4 plays a role in promoting proliferation and inhibiting apopto-sis of CRC by activating the STAT3 and ERK pathways.
    4. Discussion
    In the present study, we showed for the first time that CPA4 expres-sion is upregulated in CRC cell lines and tissue samples at both the mRNA and protein levels. Overexpression of CPA4 promoted CRC cell proliferation and colony-forming abilities, and inhibited cell apoptosis. Moreover, CPA4 also enhanced tumor growth in nude mice. In addition, knocking down CPA4 significantly inhibited CRC cell proliferation and tumor growth both in vitro and in vivo. Clinically, overexpression of CPA4, as observed by IHC staining, was associated with histological dif-ferentiation, tumor stage, and a shorter overall survival time of CRC pa-tients. These results indicated that CPA4 plays a critical role in CRC cell growth and might be a reliable biomarker for CRC.
    CPA4, also known as CPA3, is a member of the carboxypeptidase A/B subfamily. CPA4 serves as a zinc-containing exopeptidase that catalyzes the release of carboxy-terminal amino acids, and is synthesized as a zy-mogen that is activated by proteolytic cleavage [13]. Moreover, CPA4 can participate in the histone hyperacetylation pathway and may mod-ulate the function of peptides that affect the growth and regulation of prostate epithelial cells [14]. Coding variations in CPA4 may confer an increased risk of intermediate-to-high risk prostate cancer among younger patients [14]. Tanco et al. demonstrated that some of the pep-tides identified as CPA4 substrates have been previously shown to func-tion in cell differentiation and proliferation, potentially explaining the correlation between CPA4 and cancer progression [3]. Kayashima et al. reported that CPA4 is imprinted and may be a strong candidate gene for predicting prostate cancer aggressiveness [4]. Upregulation of CPA4 was also detected in the MCF-7 breast cancer cell line, and may serve as a biomarker for the prognosis of breast cancer [5]. Hsu et al. ob-served aberrant expression of CPA4 in head and neck squamous cell car-cinoma, and that this abnormality is associated with poor survival [6]. In addition, Sun et al. showed that CPA4 is overexpressed in a number of malignant tumors, and that CPA4 can exert a prognostic role in these cancers [7–12]. However, these studies did not provide insight into the functional role or underlying mechanism by which CPA4 influences cancer progression. To our knowledge, our study is the first to report the oncogenic function and mechanism of CPA4 in CRC using in vivo and in vitro experiments.
    Our study revealed that CPA4-mediated promotion of tumor cell growth may be attributable to activation of STAT3 and ERK signaling pathways. The JAK/STAT3 signaling pathway is associated with regulat-ing cell growth and proliferation, as well as reducing apoptosis, and has been reported to be activated in several types of cancer [15,16]. STAT3 activity plays an important role in various carcinogenic processes, in-cluding cell cycle regulation, prevention of apoptosis, induction of sur-vival factors, and establishment of uncontrolled growth [17,18]. The MAPK/ERK pathway is a classic intracellular signaling pathway, and is known to be closely associated with the progression of malignant tu-mors [19,20]. Accumulating evidence demonstrates that the MAPK/ ERK pathway is associated with proper performance of the cellular DNA damage response (DDR), the main pathway of tumor suppression [21]. In addition, the MAPK/ERK pathway is crucial for regulating tumor metabolism [22]. Finally, caspases are a family of protease enzymes that play essential roles in apoptosis [23,24]. We provide evidence in our study that the knockdown of CPA4 inhibits phosphorylation of the STAT3 and MAPK/ERK pathways, increasing caspase cleavage. Flow cy-tometry experiments also showed that the apoptosis index was in-creased in CPA4 knockdown cells. Moreover, after treatment with the STAT3 inhibitor cryptotanshinone or ERK inhibitor U0126 in CPA4-overexpressing cells, cell growth was suppressed and phosphorylation of STAT3 and ERK was significantly decreased. Therefore, we propose 
    that CPA4 plays an important role in tumor progression in CRC through activating the STAT3 and ERK signaling pathways. However, the direct downstream targets of CPA4 have not been identified, warranting fur-ther studies to discover the underlying molecular regulatory mechanisms.
    5. Conclusions
    Collectively, our results revealed that CPA4 was upregulated in CRC tumor samples and cell lines. Overexpression of CPA4 was positively correlated with TNM stage and poor oncological outcomes. CPA4 pro-moted CRC cell growth in vivo and in vitro through activating the STAT3 and ERK signaling pathways. Our findings highlight the onco-genic role and potential mechanism of CPA4 in promoting CRC tumorigenesis.