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Molecular Cancer Research

Poly (ADP-ribose) polymerase-1 (PARP1) is an abundant, ubiquitously expressed NAD+-dependent nuclear enzyme that has prognostic value for a multitude of human cancers. PARP1 activity serves to poly (ADP-ribose)-ylate the vast majority of known client proteins and affects a number of cellular and biologic outcomes, by mediating the DNA damage response (DDR), base-excision repair (BER), and DNA strand break (DSB) pathways. PARP1 is also critically important for the maintenance of genomic integrity, as well as chromatin dynamics and transcriptional regulation. Evidence also indicates that PARP-directed therapeutics are "synthetic lethal" in BRCA1/2-deficient model systems. Strikingly, recent studies have unearthed exciting new transcriptional-regulatory roles for PARP1, which has profound implications for human malignancies and will be reviewed herein. Mol Cancer Res; 12(8); 1069–80. ©2014 AACR.

Long noncoding RNAs (lncRNA) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor-suppressive events and lncRNAs has not been well described. Here, the novel lncRNA, prostate cancer–associated transcript 29 (PCAT29), is characterized along with its relationship to the androgen receptor. PCAT29 is suppressed by DHT and upregulated upon castration therapy in a prostate cancer xenograft model. PCAT29 knockdown significantly increased proliferation and migration of prostate cancer cells, whereas PCAT29 overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane assays. Finally, in prostate cancer patient specimens, low PCAT29 expression correlated with poor prognostic outcomes. Taken together, these data expose PCAT29 as an androgen-regulated tumor suppressor in prostate cancer.

Implications: This study identifies PCAT29 as the first androgen receptor–repressed lncRNA that functions as a tumor suppressor and that its loss may identify a subset of patients at higher risk for disease recurrence.

Visual Overview: Mol Cancer Res; 12(8); 1081–7. ©2014 AACR.

Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice than in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1+/+ mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice, but not in the other genotypes. Nuclear β-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of β-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice. Knockdown of β-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma.

Implications: This study demonstrates that inhibition of β-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4F/F;Trp53F/F;Cdh1F/+ mice, providing novel insights into mechanisms for gastric cancer metastasis. Mol Cancer Res; 12(8); 1088–99. ©2014 AACR.

Osteosarcoma is the most common primary bone malignancy and accounts for more than half of primary skeletal malignancies in children and young adults. Although vascular endothelial growth factor (VEGF) expression in osteosarcoma has been associated with poor outcome, its role in the pathogenesis of osteosarcoma remains controversial. Here, VEGF and VEGFR1 expression in both human and murine osteosarcoma cells associated with increasing malignant potential. Autocrine VEGF/VEGFR1 signaling resulted in constitutive activation of VEGFR1 in highly aggressive osteosarcoma cells. In addition, survival and proliferation of highly aggressive osteosarcoma cells was dependent on autocrine VEGF/R1 signaling in vitro. The effect of VEGFR1 expression on in vivo tumor growth and angiogenesis was evaluated by immunoselecting subpopulations of osteosarcoma cells that express high or low levels of VEGFR1. Cell enriched for high VEGFR1 expression showed increased VEGF production, tumor growth, tumor angiogenesis, and osteolysis in vivo. In addition, it was demonstrated that VEGF and VEGFR1 are coexpressed by a subset of tumor cells in human osteosarcoma, similar to what was observed in the murine osteosarcoma cells. These results suggest that autocrine VEGF/VEGFR1 signaling in a subpopulation of tumor cells plays a pivotal role in osteosarcoma progression.

Implications: Aggressive osteosarcoma phenotypes are mediated by autocrine VEGF/VEGFR1 signaling and improved stratification measures and novel anti-angiogenic strategies may benefit this specific tumor type. Mol Cancer Res; 12(8); 1100–11. ©2014 AACR.

Hypoxic conditions during the formation of colorectal cancer may support the development of more aggressive tumors. Hypoxia-inducible factor (HIF) is a heterodimeric complex, composed of oxygen-induced HIFα and constitutively expressed HIFβ subunits, which mediates the primary transcriptional response to hypoxic stress. Among HIFα isoforms, HIF1α (HIF1A) and endothelial PAS domain–containing protein 1 (EPAS1) are able to robustly activate hypoxia-responsive gene signatures. Although posttranslational regulation of HIFα subunits is well described, less is known about their transcriptional regulation. Here, molecular analysis determined that EPAS1 mRNA was significantly reduced in primary colonic adenocarcinoma specimens compared with histopathologically nonneoplastic tissue from 120 patients. In contrast, no difference in HIF1A mRNA levels was observed between cancerous and noncancerous tissue. Bisulfite DNA sequencing and high-resolution melting analysis identified significant DNA hypermethylation in the EPAS1 regulatory region from cancerous tissue compared with nonneoplastic tissue. Importantly, multivariate Cox regression analysis revealed a high HR for patients with cancer with low EPAS1 transcript levels (HR, 4.91; 95% confidence interval, CI, 0.42–56.15; P = 0.047) and hypermethylated EPAS1 DNA (HR, 33.94; 95% CI, 2.84–405.95; P = 0.0054). Treatment with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-aza-dC/Decitabine), upregulated EPAS1 expression in hypoxic colorectal cancer cells that were associated with DNA demethylation of the EPAS1 regulatory region. In summary, EPAS1 is transcriptionally regulated by DNA methylation in colorectal cancer.

Implications: DNA methylation and mRNA status of EPAS1 have novel prognostic potential for colorectal cancer. Mol Cancer Res; 12(8); 1112–27. ©2014 AACR.

PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase β (pol β) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol β–/– and Xrcc1–/– cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol β or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol β–/– and Xrcc1–/– BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency.

Implications: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy. Mol Cancer Res; 12(8); 1128–39. ©2014 AACR.

Associations of ErbB4 (ERBB4/HER4), the fourth member of the EGFR family, with cancer are variable, possibly as a result of structural diversity of this receptor. There are multiple structural isoforms of ERBB4 arising by alternative mRNA splicing, and a subset undergo proteolysis that releases membrane-anchored and soluble isoforms that associate with transcription factors and coregulators to modulate transcription. To compare the differential and common signaling activities of full-length (FL) and soluble intracellular isoforms of ERBB4, four JM-a isoforms (FL and soluble intracellular domain (ICD) CYT-1 and CYT-2) were expressed in isogenic MCF10A cells and their biologic activities were analyzed. Both FL and ICD CYT-2 promoted cell proliferation and invasion, and CYT-1 suppressed cell growth. Transcriptional profiling revealed several new and underexplored ERBB4-regulated transcripts, including: proteases/protease inhibitors (MMP3 and SERPINE2), the YAP/Hippo pathway (CTGF, CYR61, and SPARC), the mevalonate/cholesterol pathway (HMGCR, HMGCS1, LDLR, and DHCR7), and cytokines (IL8, CCL20, and CXCL1). Many of these transcripts were subsequently validated in a luminal breast cancer cell line that normally expresses ERBB4. Furthermore, ChIP-seq experiments identified ADAP1, APOE, SPARC, STMN1, and MXD1 as novel molecular targets of ERBB4. These findings clarify the diverse biologic activities of ERBB4 isoforms, and reveal new and divergent functions.

Implications: ErbB4 as a regulator of Hippo and mevalonate pathways provides new insight into milk production and anabolic processes in normal mammary epithelia and cancer. Mol Cancer Res; 12(8); 1140–55. ©2014 AACR.

FAM83B (family with sequence similarity 83, member B) was recently identified as a novel oncogene involved in activating CRAF/MAPK signaling and driving epithelial cell transformation. FAM83B is one of eight members of a protein family (FAM83) characterized by a highly conserved domain of unknown function (DUF1669), which is necessary and sufficient to drive transformation. Here, it is demonstrated that additional FAM83 members also exhibit oncogenic properties and have significantly elevated levels of expression in multiple human tumor types using a TissueScan Cancer Survey Panel PCR array and database mining. Furthermore, modeling the observed tumor expression of FAM83A, FAM83C, FAM83D, or FAM83E promoted human mammary epithelial cell (HMEC) transformation, which correlated with the ability of each FAM83 member to bind CRAF (RAF1) and promote CRAF membrane localization. Conversely, ablation of FAM83A or FAM83D from breast cancer cells resulted in diminished MAPK signaling with marked suppression of growth in vitro and tumorigenicity in vivo. Importantly, each FAM83 member was determined to be elevated in at least one of 17 distinct tumor types examined, with FAM83A, FAM83B, and FAM83D most frequently overexpressed in several diverse tissue types. Finally, evidence suggests that elevated expression of FAM83 members is associated with elevated tumor grade and decreased overall survival.

Implications: FAM83 proteins represent a novel family of oncogenes suitable for the development of cancer therapies aimed at suppressing MAPK signaling. Mol Cancer Res; 12(8); 1156–65. ©2014 AACR.

Patients with prostate cancer treated with androgen deprivation therapy (ADT) eventually develop castrate-resistant prostate cancer (CRPC). 1,25-Dihydroxyvitamin D3 (1,25D3/calcitriol) is a potential adjuvant therapy that confers antiproliferative and pro-differentiation effects in vitro, but has had mixed results in clinical trials. The impact of the tumor microenvironment on 1,25D3 therapy in patients with CRPC has not been assessed. Transforming growth factor β (TGFβ), which is associated with the development of tumorigenic "reactive stroma" in prostate cancer, induced vitamin D3 receptor (VDR) expression in the human WPMY-1 prostate stromal cell line. Similarly, TGFβ enhanced 1,25D3-induced upregulation of CYP24A1, which metabolizes 1,25D3 and thereby limits VDR activity. Ablation of Hic-5, a TGFβ-inducible nuclear receptor coregulator, inhibited basal VDR expression, 1,25D3-induced CYP24A1 expression and metabolism of 1,25D3 and TGFβ-enhanced CYP24A1 expression. A Hic-5–responsive sequence was identified upstream (392–451 bp) of the CYP24A1 transcription start site that is occupied by VDR only in the presence of Hic-5. Ectopic expression of Hic-5 sensitized LNCaP prostate tumor cells to growth-inhibitory effects of 1,25D3 independent of CYP24A1. The sensitivity of Hic-5–expressing LNCaP cells to 1,25D3-induced growth inhibition was accentuated in coculture with Hic-5–ablated WPMY-1 cells. Therefore, these findings indicate that the search for mechanisms to sensitize prostate cancer cells to the antiproliferative effects of VDR ligands needs to account for the impact of VDR activity in the tumor microenvironment.

Implications: Hic-5 acts as a coregulator with distinct effects on VDR transactivation, in prostate cancer and stromal cells, and may exert diverse effects on adjuvant therapy designed to exploit VDR activity in prostate cancer. Mol Cancer Res; 12(8); 1166–80. ©2014 AACR.

The relationship between tumor-associated macrophages (TAM) and epithelial-to-mesenchymal transition (EMT) during the initiation and progression of metastasis is still unclear. Here, a role for the vitamin D receptor (VDR) in metastasis was identified, as well as a role in the relationship between TAMs and EMT. First, the expression level of VDR was examined in clinical tissue from human patients with breast cancer or a mouse model of breast cancer with differential metastasis. These results revealed that VDR expression negatively correlates with metastasis in breast cancer. Second, coculture of VDR-overexpressing breast cancer cells with a macrophage cell line demonstrated that overexpression of VDR alleviated the prometastatic effect of cocultured macrophages on breast cancer cells. Furthermore, VDR overexpression abrogated the induction of EMT in breast cancer cells by cocultured macrophage cells, as measured by a loss of E-cadherin (CDH1) and induction of α-smooth muscle actin (α-SMA). TNFα in macrophage conditioned media inhibited VDR expression, whereas downregulation of VDR further mediated the promotion of TGFβ-induced EMT by TNFα. In addition, β-catenin expression was inhibited in VDR-overexpressing breast cancer cells and tumor xenografts. Finally, administration of calcitriol [1,25-(OH)2D3], an active vitamin D metabolite, exerted similar antimetastatic effects in breast cancer cells in vitro and a mouse model of breast cancer in vivo with preservation of VDR and suppression of β-catenin.

Implications: VDR suppression by TNFα mediates the prometastatic effect of TAMs through enhancement of the β-catenin pathway. Mol Cancer Res; 12(8); 1181–91. ©2014 AACR.