Midkine as a driver of age-related changes and increase in mammary tumorigenesis.

Aging is a pivotal risk factor for cancer, yet the underlying mechanisms remain poorly defined. Here, we explore age-related changes in the rat mammary gland by single-cell multiomics. Our findings include increased epithelial proliferation, loss of luminal identity, and decreased naive B and T cells with age. We discover a luminal progenitor population unique to old rats with profiles reflecting precancerous changes and identify midkine (Mdk) as a gene upregulated with age and a regulator of age-related luminal progenitors. Midkine treatment of young rats mimics age-related changes via activating PI3K-AKT-SREBF1 pathway and promotes nitroso-N-methylurea-induced mammary tumorigenesis. Midkine levels increase with age in human blood and mammary epithelium, and higher MDK in normal breast tissue is associated with higher breast cancer risk in younger women. Our findings reveal a link between aging and susceptibility to tumor initiation and identify midkine as a mediator of age-dependent increase in breast tumorigenesis.

Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3.

KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone-lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A-matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.

Combination Therapies to Improve the Efficacy of Immunotherapy in Triple-negative Breast Cancer.

Immune checkpoint inhibition combined with chemotherapy is currently approved as first-line treatment for patients with advanced PD-L1-positive triple-negative breast cancer (TNBC). However, a significant proportion of metastatic TNBC is PD-L1-negative and, in this population, chemotherapy alone largely remains the standard-of-care and novel therapeutic strategies are needed to improve clinical outcomes. Here, we describe a triple combination of anti-PD-L1 immune checkpoint blockade, epigenetic modulation thorough bromodomain and extra-terminal (BET) bromodomain inhibition (BBDI), and chemotherapy with paclitaxel that effectively inhibits both primary and metastatic tumor growth in two different syngeneic murine models of TNBC. Detailed cellular and molecular profiling of tumors from single and combination treatment arms revealed increased T- and B-cell infiltration and macrophage reprogramming from MHCIIlow to a MHCIIhigh phenotype in mice treated with triple combination. Triple combination also had a major impact on gene expression and chromatin profiles shifting cells to a more immunogenic and senescent state. Our results provide strong preclinical evidence to justify clinical testing of BBDI, paclitaxel, and immune checkpoint blockade combination.

Breast cancer prevention by short-term inhibition of TGFß signaling.

Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFß signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFß in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

MT4-MMP: The GPI-Anchored Membrane-Type Matrix Metalloprotease with Multiple Functions in Diseases.

MT4-MMP (or MMP17) belongs to the Membrane-Type Matrix Metalloproteinase (MT-MMP) family. This family of proteases contributes to extracellular matrix remodeling during several physiological processes, including embryogenesis, organogenesis, tissue regeneration, angiogenesis, wound healing, and inflammation. MT4-MMP (MMP17) presents unique characteristics compared to other members of the family in terms of sequence homology, substrate specificity, and internalization mode, suggesting distinct physiological and pathological functions. While the physiological functions of MT4-MMP are poorly understood, it has been involved in different pathological processes such as arthritis, cardiovascular disease, and cancer progression. The mt4-mmp transcript has been detected in a large diversity of cancers. The contribution of MT4-MMP to tumor development has been further investigated in gastric cancer, colon cancer, head and neck cancer, and more deeply in breast cancer. Given its contribution to different pathologies, particularly cancers, MT4-MMP represents an interesting therapeutic target. In this review, we examine its biological and structural properties, and we propose an overview of its physiological and pathological functions.

Expression of MT4-MMP, EGFR, and RB in Triple-Negative Breast Cancer Strongly Sensitizes Tumors to Erlotinib and Palbociclib Combination Therapy.

PURPOSE: Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB).Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. RESULTS: EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. CONCLUSIONS: We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Capecitabine Efficacy Is Correlated with TYMP and RB1 Expression in PDX Established from Triple-Negative Breast Cancers.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605-15. ©2018 AACR.

MT4-MMP and EGFR expression levels are key biomarkers for breast cancer patient response to chemotherapy and erlotinib.

BACKGROUND: Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC. METHODS: We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays. RESULTS: Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours. CONCLUSIONS: We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.