Prostate cancer (PCa) is the most common non-cutaneous cancer and second leading cause of cancer-related death for men in the United States. The nonprotein coding gene locus plasmacytoma variant translocation 1 (PVT1) is located at 8q24 and is dysregulated in different cancers. PVT1 gives rise to several alternatively spliced transcripts and microRNAs. There are at least twelve exons of PVT1, which make separate transcripts, and likely have different functions. Here, we demonstrate that PVT1 exon 9 is significantly overexpressed in PCa tissues in comparison to normal prostate tissues. Both transient and stable overexpression of PVT1 exon 9 significantly induced greater prostate epithelial cell migration, as well as increased proliferation and corresponding proliferating cell nuclear antigen (PCNA) expression. Notably, implantation into mice of a non-tumorigenic prostate epithelial cell line stably overexpressing PVT1 exon 9 resulted in the formation of malignant tumors. Furthermore, PVT1 exon 9 overexpression significantly induced castration resistance. Consequently, PVT1 exon 9 expression is important for PCa initiation and progression, and holds promise as a therapeutic target in PCa.

PVT1 is a long non-coding RNA transcribed from a gene located at the 8q24 chromosomal region that has been implicated in multiple cancers including breast cancer (BC). Amplification of the 8q24 chromosomal region is a common event in BC and is associated with poor clinical outcomes. Claudin–low (CL) triple negative breast cancer (TNBC) is a subtype of BC with a particularly dismal outcome. We assessed PVT1 exon 9 expression in the T47D estrogen receptor positive BC cell line, and in the MDA MB 468 and MDA MB 231 TNBC cell lines, followed by the assessment of the expression of claudins 1, 3, 4 and 7, in MDA MB 468 and MDA MB 231 (TNBC) cells. We found that MDA MB 231 TNBC cells significantly express less claudin 1, 3, 4, and 7 than MDA MB 468 TNBC cells. PVT1 exon 9 is significantly upregulated in MDA MB 231 CL TNBC cells, and significantly downregulated in MDA MB 468 claudin high (CH) TNBC cells, in comparison to T47D estrogen receptor positive BC cells. We then analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells. Notably, siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. Taken together, our data indicate that PVT1 exon 9 regulates claudin 4 expression and migration in CL TNBC cells, and may have clinical implications in CL TNBC.

Background: The metastasis-promoting G-protein-coupled receptor CXC Receptor 4 (CXCR4) is activated by the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1). The CXCL12/CXCR4 pathway in cancer promotes metastasis but the molecular details of how this pathway cross-talks with oncogenes are understudied. An oncogene pathway known to promote breast cancer metastasis in MDA-MB-231 xenografts is that of Mouse Double Minute 2 and 4 (MDM2 and MDM4, also known as MDMX). MDM2 and MDMX promote circulating tumor cell (CTC) formation and metastasis, and positively correlate with a high expression of CXCR4. Interestingly, this MDMX-associated upregulation of CXCR4 is only observed in cells grown in the tumor microenvironment (TME), but not in MDA-MB-231 cells grown in a tissue culture dish. This suggested a cross-talk signaling factor from the TME which was predicted to be CXCL12 and, as such, we asked if the exogenous addition of the cell non-autonomous CXCL12 ligand would recapitulate the MDMX-dependent upregulation of CXCR4. Methods: We used MDA-MB-231 cells and isolated CTCs, with and without MDMX knockdown, plus the exogenous addition of CXCL12 to determine if MDMX-dependent upregulation of CXCR4 could be recapitulated outside of the TME context. We added exogenous CXCL12 to the culture medium used for growth of MDA-MB-231 cells and isogenic cell lines engineered for MDM2 or MDMX depletion. We carried out immunoblotting, and quantitative RT-PCR to compare the expression of CXCR4, MDM2, MDMX, and AKT activation. We carried out Boyden chamber and wound healing assays to assess the influence of MDMX and CXCL12 on the cells’ migration capacity. Results: The addition of the CXCL12 chemokine to the medium increased the CXCR4 cellular protein level and activated the PI3K/AKT signaling pathway. Surprisingly, we observed that the addition of CXCL12 mediated the upregulation of MDM2 and MDMX at the protein, but not at the mRNA, level. A reduction in MDMX, but not MDM2, diminished both the CXCL12-mediated CXCR4 and MDM2 upregulation. Moreover, a reduction in both MDM2 and MDMX hindered the ability of the added CXCL12 to promote Boyden chamber-assessed cell migration. The upregulation of MDMX by CXCL12 was mediated, at least in part, by a step upstream of the proteasome pathway because CXCL12 did not increase protein stability after cycloheximide treatment, or when the proteasome pathway was blocked. Conclusions: These data demonstrate a positive feed-forward activation loop between the CXCL12/CXCR4 pathway and the MDM2/MDMX pathway. As such, MDMX expression in tumor cells may be upregulated in the primary tumor microenvironment by CXCL12 expression. Furthermore, CXCL12/CXCR4 metastatic signaling may be upregulated by the MDM2/MDMX axis. Our findings highlight a novel positive regulatory loop between CXCL12/CXCR4 signaling and MDMX to promote metastasis.

Authors: Olorunseun Ogunwobi Affiliations: Hunter College and the Graduate Center of the City University of New York Abstract Body: High mortality rates of prostate cancer (PCa) are associated with metastatic castration-resistant prostate cancer (mCRPC) due to the maintenance of androgen receptor (AR) signaling despite androgen deprivation therapies (ADTs). Resistance