osteoprotegerin and Triple-Negative-Breast-Neoplasms

Lung metastasis is one of the leading causes of death for triple-negative breast cancer (TNBC). We sought to characterize the genetic alterations underlying TNBC lung metastases by integrating whole-genome sequencing and functional screening. Furthermore, we aimed to develop a metastasis-related gene signature for TNBC patients to improve risk stratification. In this prospective observational study, we first conducted whole-genome sequencing of paired primary tumor and lung metastasis from one TNBC patient to identify potential genetic driver alterations. An in vivo gain-of-function screening using an amplified open reading frame library was then employed to screen candidate genes promoting lung metastasis. Finally, we applied Cox proportional hazard regression modeling to develop a prognostic gene signature from 14 candidate genes in TNBC. Compared to the primary tumor, copy number amplifications of chromosomes 3q and 8q were identified in the lung metastasis. We discovered an enrichment of 14 genes from chromosomes 3q and 8q in mouse lung metastases model. We further developed and validated a four-gene signature (ENY2, KCNK9, TNFRSF11B and KCNMB2) that predicts recurrence-free survival and lung metastasis in TNBC. Our data also demonstrated that upregulated expression of ENY2 could promote invasion and lung metastasis of TNBC cells both in vitro and in vivo. In conclusion, our study reveals functional genes with copy number amplifications among chromosome 3q and 8q in lung metastasis of TNBC. And we develop a functional gene signature that can effectively stratify patients into low- and high-risk subgroups of recurrence, helping frame personalized treatments for TNBC.

Topics: Animals; Biomarkers, Tumor; Cell Line, Tumor; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 8; Disease-Free Survival; DNA Copy Number Variations; Female; Follow-Up Studies; Gain of Function Mutation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Library; HEK293 Cells; Humans; Large-Conductance Calcium-Activated Potassium Channel beta Subunits; Lung Neoplasms; Middle Aged; Neoplasm Recurrence, Local; Osteoprotegerin; Potassium Channels, Tandem Pore Domain; Prognosis; Prospective Studies; Transcription Factors; Triple Negative Breast Neoplasms; Up-Regulation; Whole Genome Sequencing; Xenograft Model Antitumor Assays

Among breast cancer types, triple-negative breast cancer (TNBC) has the fewest treatment options and the lowest 5-year survival rate. Androgen receptor (AR) inhibition has displayed efficacy against breast cancer preclinically and is currently being examined clinically in AR positive TNBC patients. Androgen deprivation has been shown to induce immunogenic modulation; the alteration of tumor cell phenotype resulting in increased sensitivity to immune-mediated killing. We evaluated the ability of AR inhibition to reduce the growth and improve the immune-mediated killing of breast cancer cells with differing expression of the estrogen receptor and AR. While AR expression was required for the growth inhibitory effects of enzalutamide on breast cancer cells, both enzalutamide and abiraterone improved the sensitivity of breast cancer cells to immune-mediated lysis independent of detectable AR expression. This increase in sensitivity was linked to an increase in cell surface tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression as well as a significant reduction in the expression of osteoprotegerin (OPG). The reduction in OPG was further examined and found to be critical for the increase in sensitivity of AR- TNBC cells to immune-mediated killing. The data presented herein further support the use of AR inhibition therapy in the AR+ TNBC setting. These data, however, also support the consideration of AR inhibition therapy for the treatment of AR- TNBC, especially in combination with cancer immunotherapy, providing a potential novel therapeutic option for select patients.

Topics: Androgen Antagonists; Apoptosis; Benzamides; Biomarkers, Tumor; Cell Proliferation; Female; Humans; Immunomodulation; Immunotherapy; Nitriles; Osteoprotegerin; Phenylthiohydantoin; Receptors, Androgen; T-Lymphocytes, Cytotoxic; Triple Negative Breast Neoplasms; Tumor Cells, Cultured

Amino-bisphosphonates and statins inhibit the mevalonate pathway, and may exert anti-tumor effects. The Wnt inhibitor dickkopf-1 (DKK-1) promotes osteolytic bone lesions by inhibiting osteoblast functions and has been implicated as an adverse marker in multiple cancers. We assessed the effects of mevalonate pathway inhibition on DKK-1 expression in osteotropic breast cancer.. Regulation of DKK-1 by bisphosphonates and statins was assessed in human breast cancer cell lines, and the role of the mevalonate pathway and downstream targets was analyzed. Moreover, the potential of breast cancer cells to modulate osteoblastogenesis via DKK-1 was studied in mC2C12 cells. Clinical relevance was validated by analyzing DKK-1 expression in the tissue and serum of women with breast cancer exposed to bisphosphonates.. DKK-1 was highly expressed in receptor-negative breast cancer cell lines. Patients with receptor-negative tumors displayed elevated levels of DKK-1 at the tissue and serum level compared to healthy controls. Zoledronic acid and atorvastatin potently suppressed DKK-1 in vitro by inhibiting geranylgeranylation of CDC42 and Rho. Regulation of DKK-1 was strongest in osteolytic breast cancer cell lines with abundant DKK-1 expression. Suppression of DKK-1 inhibited the ability of breast cancer cells to block WNT3A-induced production of alkaline phosphates and bone-protective osteoprotegerin in preosteoblastic C2C12 cells. In line with the in vitro data, treatment of breast cancer patients with zoledronic acid decreased DKK-1 levels by a mean of 60% after 12 months of treatment.. DKK-1 is a novel target of the mevalonate pathway that is suppressed by zoledronic acid and atorvastatin in breast cancer.

Topics: Animals; Anticholesteremic Agents; Atorvastatin; Bone Density Conservation Agents; Bone Neoplasms; cdc42 GTP-Binding Protein; Cell Line, Tumor; Diphosphonates; Female; Gene Expression Regulation, Neoplastic; Heptanoic Acids; Humans; Imidazoles; Intercellular Signaling Peptides and Proteins; L Cells; Lymphocyte Activation; MCF-7 Cells; Mevalonic Acid; Mice; Osteoblasts; Osteogenesis; Osteoprotegerin; Prenylation; Pyrroles; Rho Factor; RNA Interference; RNA, Small Interfering; Triple Negative Breast Neoplasms; Wnt3A Protein; Zoledronic Acid

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chick Embryo; Disease Models, Animal; Disease Progression; DNA Copy Number Variations; Female; Gene Expression; Gene Knockdown Techniques; Humans; Neoplasm Metastasis; Osteoprotegerin; Patient Outcome Assessment; Peptide Hydrolases; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand; Triple Negative Breast Neoplasms