Tan A. Ince

Background: Genomic assays such as Oncotype DX, MammaPrint, and Prosigna have transformed risk stratification and treatment selection in early-stage, estrogen receptor-positive (ER+), HER2-negative breast cancers by enabling more precise prognostication and chemotherapy de-escalation in selected patients. However, their clinical utility is limited in lymph nodes positive disease. A major unmet need is the development of compact, mechanistically grounded biomarkers that extend risk and treatment-response prediction to clinically challenging ER+/HER2− subgroups, including lymph node–positive patients. Methods: Building on a cell-of-origin framework, we previously established a 70-gene triple hormone receptor (THR; ER, AR, VDR) signature (THR-70) that reflects luminal epithelial differentiation programs and is prognostic across breast cancer subtypes. Here, we refined this framework using interactome-guided clustering to derive a six-gene cell-of-origin signature (THR-6E: KIF4A, KIF2C, CDC20, FAM64A, TPX2, and LMNB2). We evaluated the prognostic and predictive performance of THR-6E across >7,000 breast cancer cases from multiple independent cohorts, assessed treatment-response prediction using endocrine- and chemotherapy-annotated datasets, and performed independent validation in the I-SPY2 adaptive clinical trial. Findings: THR-6E robustly stratifies relapse-free survival (RFS) in ER+/HER2− breast cancer independent of tumor grade, proliferation status, and subtype. Hazard ratios for RFS were 2.41 (p<1×10⁻¹⁶), 1.61 (p=4.9×10⁻⁴), and 1.50 (p=6.2×10⁻³) for grades 1, 2, and 3, respectively, and 2.16 and 1.33 for Luminal A and Luminal B subtypes. THR-6E maintained predictive value across endocrine- and chemotherapy-treated, untreated, lymph node-positive, and lymph node-negative subgroups. Beyond prognosis, THR-6E predicted endocrine therapy response in ER+/HER2−, node-negative disease and chemotherapy response in ER+/HER2−, node-positive disease, achieving approximately 70% sensitivity and specificity (AUC=0.714, p=3.6×10⁻⁶), with consistent performance across taxane-, anthracycline-, and FEC-based regimens (AUCs 0.71–0.72). Single-cell transcriptomic and proteomic analyses demonstrated that THR-6E expression is specific to normal and malignant breast glandular epithelium, preserved during transformation, and further enriched in metastatic disease. Consistent with a cell-of-origin program, THR-6E genes were rarely mutated in breast cancer and retained normal tissue-like co-expression patterns. In the I-SPY2 trial, THR-6E achieved robust prediction of pathologic complete response across multiple treatment arms (AUCs 0.72–0.94), with an overall AUC of 0.741. Interpretation: These results support a cell-of-origin–anchored approach to biomarker development and challenge purely tissue-agnostic models of therapeutic response. THR-6E represents a compact, biologically interpretable signature that extends prognostic and predictive assessment to clinically relevant ER+/HER2− subgroups, including lymph node–positive disease. Its mechanistic grounding and stable performance across cohorts position THR-6E as a complementary tool to existing multigene assays, warranting prospective diagnostic accuracy studies to define its clinical utility and impact on treatment decision-making.

Despite evidence indicating the dominance of cell-of-origin signatures in molecular tumor patterns, translating these genome-wide patterns into actionable insights has been challenging. This study introduces breast cancer cell-of-origin signatures that offer significant prognostic value across all breast cancer subtypes and various clinical cohorts, compared to previously developed genomic signatures. We previously reported that triple hormone receptor (THR) co-expression patterns of androgen (AR), estrogen (ER), and vitamin D (VDR) receptors are maintained at the protein level in human breast cancers. Here, we developed corresponding mRNA signatures (THR-50 and THR-70) based on these patterns to categorize breast tumors by their THR expression levels. The THR mRNA signatures were evaluated across 56 breast cancer datasets (5040 patients) using Kaplan–Meier survival analysis, Cox proportional hazard regression, and unsupervised clustering. The THR signatures effectively predict both overall and progression-free survival across all evaluated datasets, independent of subtype, grade, or treatment status, suggesting improvement over existing prognostic signatures. Furthermore, they delineate three distinct ER-positive breast cancer subtypes with significant survival in differences—expanding on the conventional two subtypes. Additionally, coupling THR-70 with an immune signature identifies a predominantly ER-negative breast cancer subgroup with a highly favorable prognosis, comparable to ER-positive cases, as well as an ER-negative subgroup with notably poor outcome, characterized by a 15-fold shorter survival. The THR cell-of-origin signature introduces a novel dimension to breast cancer biology, potentially serving as a robust foundation for integrating additional prognostic biomarkers. These signatures offer utility as a prognostic index for stratifying existing breast cancer subtypes and for de novo classification of breast cancer cases. Moreover, THR signatures may also hold promise in predicting hormone treatment responses targeting AR and/or VDR.

We recently identified a cell-of-origin-specific mRNA signature associated with metastasis and poor outcome in triple-negative carcinoma (TNBC). This TNBC cell-of-origin signature is associated with the over-expression of histone deacetylases and zinc finger protein HDAC1, HDAC7, and ZNF92, respectively. Based on this signature, we discovered that the combination of three drugs (an HDAC inhibitor, an anti-helminthic Niclosamide, and an antibiotic Tanespimycin that inhibits HSP90) synergistically reduces the proliferation of the twelve tested TNBC cell lines. Additionally, we discovered that four out of five inflammatory breast carcinoma cell lines are sensitive to this combination. Significantly, the concentration of the drugs that are used in these experiments are within or below clinically achievable dose, and the synergistic activity only emerged when all three drugs were combined. Our results suggest that HDAC and HSP90 inhibitors combined with the tapeworm drug Niclosamide can achieve remarkably synergistic inhibition of TNBC and IBC. Since Niclosamide, HDAC, and HSP90 inhibitors were approved for clinical use for other cancer types, it may be possible to repurpose their combination for TNBC and IBC.

Tumor phenotype is shaped both by transforming genomic alterations and the normal cell-of-origin. We identified a cell-of-origin associated prognostic gene expression signature, ET-9, that correlates with remarkably shorter overall and relapse free breast cancer survival, 8.7 and 6.2 years respectively. The genes associated with the ET-9 signature are regulated by histone deacetylase 7 (HDAC7) partly through ZNF92, a previously unexplored transcription factor with a single PubMed citation since its cloning in 1990s. Remarkably, ZNF92 is distinctively over-expressed in breast cancer compared to other tumor types, on a par with the breast cancer specificity of the estrogen receptor. Importantly, ET-9 signature appears to be independent of proliferation, and correlates with outcome in lymph-node positive, HER2+, post-chemotherapy and triple-negative breast cancers. These features distinguish ET-9 from existing breast cancer prognostic signatures that are generally related to proliferation and correlate with outcome in lymph-node negative, ER-positive, HER2-negative breast cancers. Our results suggest that ET-9 could be also utilized as a predictive signature to select patients for HDAC inhibitor treatment.

The angiogenic factor, VEGFA, is a therapeutic target in ovarian cancer (OVCA). VEGFA can also stimulate stem‐like cells in certain cancers, but mechanisms thereof are poorly understood. Here, we show that VEGFA mediates stem cell actions in primary human OVCA culture and OVCA lines via VEGFR2‐dependent Src activation to upregulate Bmi1, tumor spheres, and ALDH1 activity. The VEGFA‐mediated increase in spheres was abrogated by Src inhibition or SRC knockdown. VEGFA stimulated sphere formation only in the ALDH1⁺ subpopulation and increased OVCA‐initiating cells and tumor formation in vivo through Bmi1. In contrast to its action in hemopoietic malignancies, DNA methyl transferase 3A (DNMT3A) appears to play a pro‐oncogenic role in ovarian cancer. VEGFA‐driven Src increased DNMT3A leading to miR‐128‐2 methylation and upregulation of Bmi1 to increase stem‐like cells. SRC knockdown was rescued by antagomir to miR‐128. DNMT3A knockdown prevented VEGFA‐driven miR‐128‐2 loss, and the increase in Bmi1 and tumor spheres. Analysis of over 1,300 primary human OVCAs revealed an aggressive subset in which high VEGFA is associated with miR‐128‐2 loss. Thus, VEGFA stimulates OVCA stem‐like cells through Src‐DNMT3A‐driven miR‐128‐2 methylation and Bmi1 upregulation. Synopsis VEGFA is not only a key mediator of angiogenesis during tumor progression, but can also act to expand the population of ovarian cancer‐initiating cells with stem‐like properties. VEGFA increases sphere formation and ALDH1 activity of ovarian cancer cells in vitro and stimulates ovarian tumor‐initiating cells in vivo. VEGFA rapidly activates VEGFR2 and Src to upregulate DNMT3 expression. DNMT3A plays a pro‐oncogenic role to methylate miR‐128‐2 leading to Bmi1 and OVCA stem‐like cell upregulation. High VEGFA and decreased miR‐128‐2 associate with poor ovarian cancer outcome.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell–like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment. Cancer Res; 76(2); 491–504. ©2016 AACR.