JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer.

INTRODUCTION: We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer. METHODS: Through microarray informatics, Cox proportional hazards regression was used to analyze the correlation between gene expression and distant metastasis-free survival (DMFS) of patients in 14 independent breast cancer cohorts. JMJD6 emerged as a top candidate gene robustly associated with poor patient survival. Immunohistochemistry, siRNA-mediated silencing, and forced overexpression of JMJD6 in cell-based assays elucidated molecular mechanisms of JMJD6 action in breast cancer progression and shed light on the clinical breast cancer subtypes relevant to JMJD6 action. RESULTS: JMJD6 was expressed at highest levels in tumors associated with worse outcomes, including ER- and basal-like, Claudin-low, Her2-enriched, and ER+ Luminal B tumors. High nuclear JMJD6 protein was associated with ER negativity, advanced grade, and poor differentiation in tissue microarrays. Separation of ER+/LN- patients that received endocrine monotherapy indicated that JMJD6 is predictive of poor outcome in treatment-specific subgroups. In breast cancer cell lines, loss of JMJD6 consistently resulted in suppressed proliferation but not apoptosis, whereas forced stable overexpression increased growth. In addition, knockdown of JMJD6 in invasive cell lines, such as MDA-MB231, decreased motility and invasion, whereas overexpression in MCF-7 cells slightly promoted motility but did not confer invasive growth. Microarray analysis showed that the most significant transcriptional changes occurred in cell-proliferation genes and genes of the TGF-ß tumor-suppressor pathway. High proliferation was characterized by constitutively high cyclin E protein levels. The inverse relation of JMJD6 expression with TGF-ß2 could be extrapolated to the breast cancer cohorts, suggesting that JMJD6 may affect similar pathways in primary breast cancer. CONCLUSIONS: JMJD6 is a novel biomarker of tumor aggressiveness with functional implications in breast cancer growth and migration.

Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer.

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

RCP is a human breast cancer-promoting gene with Ras-activating function.

Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.

Frequent decreased expression of candidate tumor suppressor gene, DEC1, and its anchorage-independent growth properties and impact on global gene expression in esophageal carcinoma.

Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development.