Abstract

Background

   Cancer stem cells (CSCs) are a small subpopulation of cells within
   tumors with stem cell property. Increased evidence suggest that CSCs
   could be responsible for chemoresistance and recurrence in colorectal
   cancer (CRC). However, a reliable therapeutic target on CSCs is still
   lacking.

Methods

   Here we describe a two-step strategy to generate CSC targets with high
   selectivity for colon stem cell markers, specific proteins that are
   interacted with CSC markers were selected and subsequently validated in
   a survival analysis. TMEM17 protein was found and its biological
   functions in CRC cells were further examined. Finally, we utilized the
   Gene Set Enrichment Analysis (GSEA) to investigate the potential
   mechanisms of TMEM17 in CRC.

Results

   By combining protein–protein interaction (PPI) database and
   high-throughput gene profiles, network analysis revealed a cluster of
   colon CSCs related genes. In the cluster, TMEM17 was identified as a
   novel CSCs related gene. The results of in-vitro functional study
   demonstrated that TMEM17 depletion can suppress the proliferation of
   CRC cells and sensitize CRC cells to chemotherapy drugs. Enrichment
   analysis revealed that the expression of TMEM17 is associated with the
   magnitude of activation of the Wnt/β-catenin pathway. Further
   validation in clinical samples demonstrated that the TMEM17 expression
   was much higher in tumor than normal tissue and was associated with
   poor survival in CRC patients.

Conclusion

   Collectively, our finding unveils the critical role of TMEM17 in CRC
   and TMEM17 could be a potential effective therapeutic target for tumor
   recurrence and chemoresistance in the colorectal cancer (CRC).

   Keywords: Colorectal cancer, Cancer stem cell, TMEM17, Chemoresistance,
   Protein–protein interaction

Introduction

   Colorectal cancer (CRC) is one of the most common cancer types in the
   world. A recent statistic study demonstrated that over one million new
   cases of CRC were identified in 2018 globally [[42]1]. Although
   advances in the treatments of CRC improved the disease outcome, the
   tumor recurrence and chemoresistance remain major causes of therapy
   failure [[43]2, [44]3]. A highly treatment resistant core portion of
   cancer cells, termed “cancer stem cells” (CSCs), could be responsible
   for these treatment failures. In line with this hypothesis, studies
   have discovered stem cell markers including LGR5, CD24, CD44, EPCAM,
   CD133 on a small proportion of CRC population [[45]4–[46]11]. In
   addition, this highly chemotherapy resistant population of CSCs
   exhibits stem cell properties including self-renewal and generation of
   mature differentiated cancer cells [[47]12, [48]13]. These findings
   suggest that the CSC population could be a potential target for CRC
   treatment, its application value in clinic need to be examined.

   Since abundant amount of patients’ genomic profiles has been taken up
   in public data sets, which are free of access, the high-throughput data
   analysis has emerged as a new efficient and cost saving method for the
   cancer research [[49]14]. In this study, we found a cluster of cancer
   stem cell related proteins using a systematic and protein interaction
   network analysis. Among these proteins, TMEM17 is closely related to
   the colon cancer stem cell markers. TMEM17 belongs to the transmembrane
   (TMEM) protein family, which is involved in numerous pathological
   processes of the cancer development, such as local invasion, metastasis
   formation and intravasation [[50]15, [51]16]. Diverse functions of
   TMEM17 family member have been reported, for example TMEM48, TMEM45A
   and TMEM97 were reported as potential prognostic biomarkers for
   cancers, TMEM16A was found related to calcium regulation and TMEM173
   was reported to control the immune response in carcinogenesis
   [[52]16–[53]18].

   To our knowledge, just a few studies have shown that TMEM17 is
   associated with the cancer development. One claimed that TMEM17 is a
   pro-oncogenic protein in the breast cancer, while another declared that
   TMEM17 is an anti-oncogenic protein in the lung cancer [[54]19,
   [55]20]. It is still unknown why TMEM17 demonstrated these contradict
   effects and what is its effect on other type of cancers such as CRC. In
   this study, we describe a two-step strategy to generate CSC targets
   with high selectivity for colon stem cell markers, specific proteins
   associated with CSC markers were selected and subsequently validated in
   a survival analysis. TMEM17 was found in the selecting process and its
   biological functions was further investigated in CRC cell lines. The
   results demonstrated that depletion of TMEM17 enhances the sensitivity
   of chemotherapy drugs and suppresses the Wnt/β-catenin signaling. Taken
   together, our study identifies an important role of TMEM17 in colon
   cancer and elucidates a potential cancer stem cell target to sensitize
   chemotherapy.

Methods

Patients

   We retrospectively analyzed the gene expression profiles of frozen
   colorectal cancer tumor tissue samples from one of the largest
   individual data sets: CIT/[56]GSE39582 CRC cohort. The data set was
   obtained directly in its processed format from GEO database through
   Bioconductor package ‘GEOquery’. All patients were included in this
   study. The batch effects were corrected using ‘combat’ algorithm
   implemented in R package ‘sva’ and z-scores for each gene were used for
   the following analyses. Both paper charts and electronic medical
   records were carefully reviewed when necessary.

Construction and analysis of protein–protein interaction network

   To find potential therapeutic targets on colon CSCs, nine colon stem
   cell markers selected from previous studies were used to construct a
   protein–protein interaction (PPI) network related to colon CSCs (Table
   [57]1). The protein interaction information of these proteins were
   obtained from the BioGRID database (Version 3.5.168) [[58]21, [59]22].
   To investigate the association of chemotherapeutic sensitivity, 232
   patients with chemotherapy and complete prognostic information in the
   CIT cohort ([60]GSE39582) were used as the discovery data set [[61]23].
   To obtain genes related to prognosis of colorectal cancer and avoid the
   influence of sample distribution, the corresponding genes resulted from
   PPI analysis were further examined using the log-rank test with 1000
   times randomization (80% portion of samples each time) to assess the
   association between each gene and patients’ disease-free survival in
   the discovery cohort. Genes with significant frequency found in more
   than 500 times in repeated log-rank tests were identified as key genes.

Table 1.

   List of colon cancer stem cell markers
   Gene Other name Function References