Background
CBX7 belongs to the polycomb repressive complex 1 (PRC1), a multiprotein complex that together with the polycomb repressive complex 2 (PRC2) inhibits the transcription of the developmental genes [
1‐
3]. A crucial activity of CBX7 in tumor progression is supported by several studies [
4]. Indeed, a drastic downregulation of CBX7 expression has been described in thyroid [
5], pancreatic [
6], colon [
7], lung [
8], gastric [
9], bladder [
10], breast [
11] carcinomas, and a more advanced stage of neoplastic disease and a poor survival has been directly correlated to the loss of
CBX7 expression [
6,
7]. Furthermore, when
CBX7 expression is restored in thyroid [
5], gastric [
9] and colon [
7] carcinoma cells there is a decreased proliferation rate with the accumulation of the cells in the G1 phase of the cell cycle, suggesting a negative role of CBX7 on the control of cell growth and, particularly, in the regulation of the G1/S switch of the cell cycle [
5].
CBX7 is able to interact with different proteins, modulating in positive or negative the expression of several genes implicated in various biological functions [
4]. In particular, it positively regulates the expression of E-cadherin [
12] that is required to maintain the regular morphology of epithelial cell, and whose loss of expression is associated with the epithelial-mesenchymal transition [
13,
14]. The CBX7 activation of E-cadherin expression is caused by the interaction with histone deacetylase 2 and inhibition of its action on the CDH1 promoter [
12]. Consistently, two studies, in thyroid [
12] and pancreatic [
6] carcinomas, report a direct correlation between the
CBX7 expression and the E-cadherin levels.
Moreover, we have also shown that CBX7 counteracts the HMGA-induced activation of the
SPP1 gene [
15], encoding the chemokine osteopontin, that is highly overexpressed in several human carcinomas and has a key function in malignant transformation. Furthermore, CBX7 inhibits, by a similar mechanism, the promoter activity of Cyclin E gene [
8], that enhances the transition of the G1 to S phase of the cell cycle then increasing the cell proliferation rate. Therefore, on the basis of all these studies, the absence of the
CBX7 gene expression plays an important function in the late stages of human malignancies [
4].
MicroRNAs (miRNAs) have become known as a significant class of short endogenous RNAs that control gene expression at post-transcriptional level through base-pairing with their target mRNAs for direct cleavage or by inhibiting mRNA translation [
16‐
18]. They have a central function in a lot of biological pathways, as developmental process, signaling transduction, stem cell differentiation, cell growth, and cancer [
19,
20].
In the current study, we have carried out an analysis of miRNA expression profiling in mouse embryonic fibroblasts (MEFs) obtained from Cbx7-knockout (KO) and wild-type (WT) mice to search for the miRNAs modulated by the Cbx7 protein. Among the differentially expressed miRNAs in WT and Cbx7-null MEFs, we concentrated our attention on the miR-155, downregulated in homozygous Cbx7-KO MEFs in comparison to the WT. Consistently, we report downregulation of both CBX7 and miR-155 expression in a set of colon carcinomas. Finally, we demonstrate that KRAS gene is a target of miR-155.
Discussion
CBX7 has been already proposed as tumor suppressor gene, since several studies have demonstrated that its expression is drastically downregulated in most of the malignant neoplasias [
4], and the development of liver and lung carcinomas in
Cbx-7 null mice further supports the CBX7 tumor suppressor activity [
8]. Moreover, the restoration of
CBX7 expression in carcinoma cells of different origin results into a reduced growth rate [
5,
7,
11,
33], blocking the cells in the G1 phase of the cell cycle. Very recently, we have also reported that reestablishment of
CBX7 expression in two cell lines of human lung carcinoma, in which it was undetectable, yields a diminished proliferation and an improved apoptosis after drug exposure [
21]. However, CBX7 has been reported to exert also oncogenic activity. Indeed, it was shown that
Cbx7 expression in mice lymphoid compartment can promote T cell lymphomagenesis and, working together with c-Myc, produces aggressive B cell lymphomas by downregulating Ink4a/Arf locus [
1]. Equally, CBX7-positive patients affected by ovarian carcinoma showed significantly shorter overall and progression-free survival rates than those of the CBX7-negative patients. Moreover,
CBX7 knockdown significantly reduced cell viability in two ovarian carcinoma cell lines compared to the control cells, likely by the upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [
34]. Recently, we have reported that CBX7 regulates several genes involved in tumor progression [
12,
35] such as E cadherin, cyclin E,
SPP1 likely accounting for the critical role of CBX7 in carcinogenesis.
In this study, we have envisaged the hypothesis that CBX7 tumor suppressor or oncogenic role may be also mediated by the regulation of miRNA expression since the role of miRNAs in cancer development and progression has been frequently reported [
36]. Consistently, we previously demonstrated that CBX7 negatively regulates the expression of miR-181 that has among its targets CBX7, creating a synergistic loop that contributes to breast cancer progression [
11].
Then, we have studied the miRNA expression profile of Cbx7 null MEFs versus the wt. Nine miRNAs were found downregulated and twenty upregulated with a fold change higher than 2 in the Cbx7 KO MEFs with respect to WT counterparts. We first validated the results of the array analyzing, by qRT-PCR, 5 deregulated miRNAs in Cbx7 KO and WT MEFs.
On the basis of the opposite functions of CBX7, as oncogene and oncosuppressor, it was not surprising to find that CBX7 was able to regulate in opposite sense miRNAs that have recognized to have oncogenic functions, such as miR-199 (negatively regulated) and miR-155, miR-221 and miR-222 (positively regulated). Equally, we observe that a potential oncosuppressor gene, such as miR-137, is regulated negatively, whereas another one, such as miR-323, is regulated positively. It is also not unlikely that the modulation of these miRNAs might be specific of MEFs and that CBX7 could regulate the same miRNA positively or negatively depending on the cellular context. Therefore, we retain that the recent results evidencing a role of the loss of CBX7 expression in the progression of human colon cancer might be also explained by the ability of CBX7 to modulate the expression of several miRNAs.
Subsequently, we decided to focus on the miR-155 since it was one of the most downregulated miRNAs in
Cbx7 KO MEFs. Moreover, miR-155 is the most commonly miRNA overexpressed in malignancies among the cancer-related miRNAs [
28]. Bcl6, HDAC4, msh2, msh6, mlh1 have been already identified as targets of miR-155 [
26]. In liposarcoma casein kinase 1-α (CK1-α) is targeted by miR-155, enhancing beta-catenin and cyclin D1 [
27]. In this study we have identified
KRAS as target of miR-155, since overexpression of miR-155 leads to a drastic reduction of the
KRAS mRNA and protein levels indicating an effect of miR-155 also on
KRAS mRNA degradation. Consistently, higher KRAS protein levels were detected in
Cbx7 null MEFs in comparison with the WT. The same result was achieved when
CBX7 expression was restored in lung carcinoma cells. However, the analysis of KRAS specific mRNA does not reveal any correlation with miR-155 and CBX7 expression. Maybe this correlation is present at protein levels, and we can also hypothesize that the regulation of KRAS by miR-155 is specific of some tissues depending on the cellular context.
It is likely that the targeting of
KRAS may have a role in the anti-apoptotic activity of miR-155 observed in monocytic differentiation where miR-155 seems to have as targets other anti-apoptotic factors such as RPS6KA3, SGK3, and RHEB. It is likely that this anti-apoptotic activity may be important for the growth of MEFs, also associated to the increased mR-221 and miR-222 levels that are able to target p27 [
37], a critical negative regulator of the cell cycle.
Interestingly, CBX7 seems to positively regulate miR-323: this can account for the reduced miR-323 expression in prostate cancer where CBX7 is drastically downregulated [
38] and miR-323 has a tumor suppressor activity by targeting AdipoRI [
39].
Acknowledgements
Dr Pierlorenzo Pallante for his helpful supports.