No abstract

Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies.

Loss of IQGAP2 contributes to the tumorigenesis of hepatocellular carcinoma and gastric cancer. However, whether IQGAP2 also suppresses prostate tumorigenesis remains unclear. We report here that IQGAP2 is a candidate tumour suppressor of prostate cancer (PC). Elevated IQGAP2 was detected in prostatic intraepithelial neoplasia (PIN), early stages of PCs (Gleason score ≤3), and androgen-dependent LNCaP PC cells. However, IQGAP2 was expressed at substantially reduced levels not only in prostate glands and non-tumorigenic BPH-1 prostate epithelial cells but also in advanced (Gleason score 4 or 5) and androgen-independent PCs. Furthermore, xenograft tumours that were derived from stem-like DU145 cells displayed advanced features and lower levels of IQGAP2 in comparison to xenograft tumours that were produced from non stem-like DU145 cells. Collectively, these results suggest that IQGAP2 functions in the surveillance of prostate tumorigenesis. Consistent with this concept, ectopic IQGAP2 reduced the proliferation of DU145, PC3, and 293T cells as well as the invasion ability of DU145 cells. While ectopic IQGAP2 up-regulated E-cadherin in DU145 and PC3 cells, knockdown of IQGAP2 reduced E-cadherin expression. In primary PC and DU145 cells-derived xenograft tumours, the majority of tumours with high levels of IQGAP2 were strongly-positive for E-cadherin. Therefore, IQGAP2 may suppress PC tumorigenesis, at least in part, by up-regulation of E-cadherin. Mechanistically, overexpression of IQGAP2 significantly reduced AKT activation in DU145 cells and inhibition of AKT activation upregulated E-cadherin, suggesting that IQGAP2 increases E-cadherin expression by inhibiting AKT activation. Taken together, we demonstrate here that IQGAP2 is a candidate tumour suppressor of PC.

Background:Accumulating evidence demonstrates high levels of aldehyde dehydrogense (ALDH) activity in human cancer types, in part, because of its association with cancer stem cells. Whereas ALDH1A1 and ALDH7A1 isoforms were reported to associate with prostate tumorigenesis, whether other ALDH isoforms are associated with prostate cancer (PC) remains unclear.Methods:ALDH3A1 expression was analysed in various PC cell lines. Xenograft tumours and 54 primary and metastatic PC tumours were stained using immunohistochemistry for ALDH3A1 expression.Results:In comparison with the non-stem counterparts, a robust upregulation of ALDH3A1 was observed in DU145-derived PC stem cells (PCSCs). As DU145 PCSCs produced xenograft tumours with more advanced features compared with those derived from DU145 cells, higher levels of ALDH3A1 were detected in the former; a dramatic elevation of ALDH3A1 occurred in DU145 cell-derived lung metastasis compared with local xenograft tumours. Furthermore, while ALDH3A1 was not observed in prostate glands, ALDH3A1 was clearly present in PIN, and further increased in carcinomas. In comparison with the paired local carcinomas, ALDH3A1 was upregulated in lymph node metastatic tumours; the presence of ALDH3A1 in bone metastatic PC was also demonstrated.Conclusions:We report here the association of ALDH3A1 with PC progression.

The BMI1 protein contributes to stem cell pluripotency and oncogenesis via multiple functions, including its newly identified role in DNA damage response (DDR). Although evidence clearly demonstrates that BMI1 facilitates the repair of double-stranded breaks via homologous recombination (HR), it remains unclear how BMI1 regulates checkpoint activation during DDR. We report here that BMI1 has a role in G2/M checkpoint activation in response to etoposide (ETOP) treatment. Ectopic expression of BMI1 in MCF7 breast cancer and DU145 prostate cancer cells significantly reduced ETOP-induced G2/M arrest. Conversely, knockdown of BMI1 in both lines enhanced the arrest. Consistent with ETOP-induced activation of the G2/M checkpoints via the ATM pathway, overexpression and knockdown of BMI1, respectively, reduced and enhanced ETOP-induced phosphorylation of ATM at serine 1981 (ATM pS1981). Furthermore, the phosphorylation of ATM targets, including γH2AX, threonine 68 (T68) on CHK2 (CHK2 pT68) and serine 15 (S15) on p53 were decreased in overexpression and increased in knockdown BMI1 cells in response to ETOP. In line with the requirement of NBS1 in ATM activation, we were able to show that BMI1 associates with NBS1 and that this interaction altered the binding of NBS1 with ATM. BMI1 consists of a ring finger (RF), helix-turn-helix-turn-helix-turn (HT), proline/serine (PS) domain and two nuclear localization signals (NLS). Although deletion of either RF or HT did not affect the association of BMI1 with NBS1, the individual deletions of PS and one NLS (KRMK) robustly reduced the interaction. Stable expression of these BMI1 mutants decreased ETOP-induced ATM pS1981 and CHK2 pT68, but not ETOP-elicited γH2AX in MCF7 cells. Furthermore, ectopic expression of BMI1 in non-transformed breast epithelial MCF10A cells also compromised ETOP-initiated ATM pS1981 and γH2AX. Taken together, we provide compelling evidence that BMI1 decreases ETOP-induced G2/M checkpoint activation via reducing NBS1-mediated ATM activation.

Contactin-1 has been shown to promote cancer metastasis. However, the underlying mechanisms remain unclear. We report here that knockdown of contactin-1 in A549 lung cancer cells reduced A549 cell invasion and the cell's ability to grow in soft agar without affecting cell proliferation. Reduction of contactin-1 resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. In an effort to investigate the mechanisms whereby contactin-1 reduces E-cadherin expression, we observed that contactin-1 plays a role in AKT activation, as knockdown of contactin-1 attenuated AKT activation. Additionally, inhibition of AKT activation significantly enhanced E-cadherin expression, an observation that mimics the situation observed in contactin-1 knockdown, suggesting that activation of AKT plays a role in contactin-1-mediated downregulation of E-cadherin. In addition, we were able to show that knockdown of contactin-1 did not further reduce A549 cell's invasion ability, when AKT activation was inhibited by an AKT inhibitor. To further support our findings, we overexpressed CNTN-1 in two CNTN-1 null breast cancer cell lines expressing E-cadherin. Upon overexpression, CNTN-1 reduced E-cadherin levels in one cell line and increased AKT activation in the other. Furthermore, in our study of 63 primary lung cancers, we observed 65% of primary lung cancers being contactin-1 positive and in these carcinomas, 61% were E-cadherin negative. Collectively, we provide evidence that contactin-1 plays a role in the downregulation of E-cadherin in lung cancer and that AKT activation contributes to this process. In a study of mechanisms responsible for contactin-1 to activate AKT, we demonstrated that knockdown of CNTN-1 in A549 cells did not enhance PTEN expression but upregulated PHLPP2, a phosphatase that dephosphorylates AKT. These observations thus suggest that contactin-1 enhances AKT activation in part by preventing PHLPP2-mediated AKT dephosphrorylation.

Although the FAM84B gene lies within chromosome 8q24, a locus frequently altered in prostate cancer (PC), its alteration during prostate tumorigenesis has not been well studied. We report here FAM84B upregulation in DU145 cell-derived prostate cancer stem-like cells (PCSLCs) and DU145 cell-produced lung metastases compared to subcutaneous xenograft tumors. FAM84B protein was detected in bone metastases and primary PCs. Nanostring examination of 7 pairs of tumor adjacent normal and PC tissues revealed elevations in FAM84B mRNA levels in all carcinomas. Furthermore, through analysis of FAM84B expression using large datasets within the Gene Expression Omnibus and OncomineTM database, we demonstrate significant increases in FAM84B mRNA in 343 primary PCs versus 181 normal tissues, and elevations in the FAM84B gene copy number (GCN) in 171 primary PCs versus 61 normal tissues. While FAM84B was not detected at higher levels via immunohistochemistry in high grade (Gleason score/GS 8-10) tumors compared to GS6-7 PCs, analyses of FAM84B mRNA and GCN using datasets within the cBioPortal database demonstrated FAM84B upregulation in 12% (67/549) of primary PCs and 18% (73/412) of metastatic castration resistant PCs (mCRPCs), and GCN increases in 4.8% (26/546) of primary PCs and 26% (121/467) of mCRPCs, revealing an association of the aforementioned changes with CRPC development. Of note, an increase in FAM84B expression was observed in xenograft CRPCs produced by LNCaP cells. Furthermore, FAM84B upregulation and GCN increases correlate with decreases in disease free survival and overall survival. Collectively, we demonstrate a novel association of FAM84B with PC tumorigenesis and CRPC progression.

Evidence supports the upregulation of MUC1 in prostate cancer (PC). However, this has not been thoroughly investigated. We report here an association of MUC1 upregulation with PC metastasis and the development of castration resistant PC (CRPC). MUC1 expression was specifically increased in DU145 cell-derived PC stem-like cells (PCSLCs) in comparison to their non-PCSLCs counterparts. While immunohistochemistry staining of 34 primary PCs revealed variability in MUC1 expression, Nanostring technology demonstrated elevated MUC1 mRNA levels in 4 of 7 PCs compared to their normal matched tissues. By analyzing MUC1 mRNA levels and gene copy number (GCN) using the OncomineTM database, elevations in MUC1 mRNA in 82 metastases versus 280 primary PCs and in MUC1 GCN in 37 metastases over 181 primary tumors were demonstrated. Analysis of genomic datasets within cBioPortal revealed increases in MUC1 GCN in 2% (6/333) of primary PCs, 6% (9/150) of metastatic PCs, and 33% (27/82) of CRPCs; in comparison, the respective increase in androgen receptor (AR) GCN was 1%, 63%, and 56%, revealing a specific increase in MUC1 GCN for CRPC. Furthermore, a 25-gene MUC1 network was amplified in 52% of CRPCs compared to 69% of CRPCs displaying increases in an AR co-regulator group. While genomic alterations in the MUC1 network largely overlap with those in the AR group, 18 CRPCs (66.7% being neuroendocrine PC) showed genomic alterations only in the MUC1 network. Moreover, genomic alterations in the MUC1 network correlated with PC relapse. Collectively, our observations suggest a combination therapy involving MUC1-based immunotherapy and androgen deprivation.