Jamie C. Stanford

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

HER3 Is Required for HER2-Induced Preneoplastic Changes to the Breast Epithelium and Tumor Formation

Increasing evidence suggests that HER2-amplified breast cancer cells use HER3/ErbB3 to drive therapeutic resistance to HER2 inhibitors. However, the role of ErbB3 in the earliest events of breast epithelial transformation remains unknown. Using mouse mammary specific models of Cre-mediated ErbB3 ablation, we show that ErbB3 loss prevents the progressive transformation of HER2-overexpressing mammary epithelium. Decreased proliferation and increased apoptosis were seen in MMTV-HER2 and MMTV-Neu mammary glands lacking ErbB3, thus inhibiting premalignant HER2-induced hyperplasia. Using a transgenic model in which HER2 and Cre are expressed from a single polycistronic transcript, we showed that palpable tumor penetrance decreased from 93.3% to 6.7% upon ErbB3 ablation. Penetrance of ductal carcinomas in situ was also decreased. In addition, loss of ErbB3 impaired Akt and p44/42 phosphorylation in preneoplastic HER2-overexpressing mammary glands and in tumors, decreased growth of preexisting HER2-overexpressing tumors, and improved tumor response to the HER2 tyrosine kinase inhibitor lapatinib. These events were rescued by reexpression of ErbB3, but were only partially rescued by ErbB36F, an ErbB3 mutant harboring six tyrosine-to-phenylalanine mutations that block its interaction with phosphatidyl inositol 3-kinase. Taken together, our findings suggest that ErbB3 promotes HER2-induced changes in the breast epithelium before, during, and after tumor formation. These results may have important translational implications for the treatment and prevention of HER2-amplified breast tumors through ErbB3 inhibition.

MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK-/- mice. Transplantation of MerTK-/- bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b+ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK-/- leukocytes exhibited lower tumor cell-induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8+ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK-/- mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8+ T lymphocyte depletion restored tumor growth in MerTK-/- mice. These data demonstrate that MerTK signaling in tumor-associated CD11b+ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.

ErbB3 Ablation Impairs PI3K/Akt-Dependent Mammary Tumorigenesis

The ErbB receptor family member ErbB3 has been implicated in breast cancer growth, but it has yet to be determined whether its disruption is therapeutically valuable. In a mouse model of mammary carcinoma driven by the polyomavirus middle T (PyVmT) oncogene, the ErbB2 tyrosine kinase inhibitor lapatinib reduced the activation of ErbB3 and Akt as well as tumor cell growth. In this phosphatidylinositol-3 kinase (PI3K)-dependent tumor model, ErbB2 is part of a complex containing PyVmT, p85 (PI3K), and ErbB3, that is disrupted by treatment with lapatinib. Thus, full engagement of PI3K/Akt by ErbB2 in this oncogene-induced mouse tumor model may involve its ability to dimerize with and phosphorylate ErbB3, which itself directly binds PI3K. In this article, we report that ErbB3 is critical for PI3K/Akt-driven tumor formation triggered by the PyVmT oncogene. Tissue-specific, Cre-mediated deletion of ErbB3 reduced Akt phosphorylation, primary tumor growth, and pulmonary metastasis. Furthermore, EZN-3920, a chemically stabilized antisense oligonucleotide that targets the ErbB3 mRNA in vivo, produced similar effects while causing no toxicity in the mouse model. Our findings offer further preclinical evidence that ErbB3 ablation may be therapeutically effective in tumors where ErbB3 engages PI3K/Akt signaling.

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution

Breast cancers that occur in women 2-5 years postpartum are more frequently diagnosed at metastatic stages and correlate with poorer outcomes compared with breast cancers diagnosed in young, premenopausal women. The molecular mechanisms underlying the malignant severity associated with postpartum breast cancers (ppBCs) are unclear but relate to stromal wound-healing events during postpartum involution, a dynamic process characterized by widespread cell death in milk-producing mammary epithelial cells (MECs). Using both spontaneous and allografted mammary tumors in fully immune-competent mice, we discovered that postpartum involution increases mammary tumor metastasis. Cell death was widespread, not only occurring in MECs but also in tumor epithelium. Dying tumor cells were cleared through receptor tyrosine kinase MerTK-dependent efferocytosis, which robustly induced the transcription of genes encoding wound-healing cytokines, including IL-4, IL-10, IL-13, and TGF-β. Animals lacking MerTK and animals treated with a MerTK inhibitor exhibited impaired efferocytosis in postpartum tumors, a reduction of M2-like macrophages but no change in total macrophage levels, decreased TGF-β expression, and a reduction of postpartum tumor metastasis that was similar to the metastasis frequencies observed in nulliparous mice. Moreover, TGF-β blockade reduced postpartum tumor metastasis. These data suggest that widespread cell death during postpartum involution triggers efferocytosis-induced wound-healing cytokines in the tumor microenvironment that promote metastatic tumor progression.

ErbB3 downregulation enhances luminal breast tumor response to antiestrogens

Aberrant regulation of the erythroblastosis oncogene B (ErbB) family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers. ErbB3 is required in luminal mammary epithelial cells (MECs) for growth and survival. Since breast cancer phenotypes may reflect biological traits of the MECs from which they originate, we tested the hypothesis that ErbB3 drives luminal breast cancer growth. We found higher ERBB3 expression and more frequent ERBB3 gene copy gains in luminal A/B breast cancers compared with other breast cancer subtypes. In cell culture, ErbB3 increased growth of luminal breast cancer cells. Targeted depletion of ErbB3 with an anti-ErbB3 antibody decreased 3D colony growth, increased apoptosis, and decreased tumor growth in vivo. Treatment of clinical breast tumors with the antiendocrine drug fulvestrant resulted in increased ErbB3 expression and PI3K/mTOR signaling. Depletion of ErbB3 in fulvestrant-treated tumor cells reduced PI3K/mTOR signaling, thus decreasing tumor cell survival and tumor growth. Fulvestrant treatment increased phosphorylation of all ErbB family RTKs; however, phospho-RTK upregulation was not seen in tumors treated with both fulvestrant and anti-ErbB3. These data indicate that upregulation of ErbB3 in luminal breast cancer cells promotes growth, survival, and resistance to fulvestrant, thus suggesting ErbB3 as a target for breast cancer treatment.

Efferocytosis creates a tumor microenvironment supportive of tumor survival and metastasis

Programmed cell death, or apoptosis, occurs in nearly all tissues of all multi-cellular organisms. In order to avoid leakage of intracellular contents, which could generate tissue damaging inflammation, apoptotic cells are cleared from tissues by phagocytes, which then dispatch the engulfed dying cell through the lysosomal pathway. Phagocytic clearance of apoptotic cells is referred to as efferocytosis. One key feature of efferocytosis is the production and release of wound healing cytokines by the phagocyte, which acts to resolve inflammation, and promote tissue repair. Phagocytic engulfment of apoptotic cells coupled with cytokine modulation aimed at immune suppression ensures that physiological programmed cell death does not induce inflammation and tissue damage. However, cytokines involved in wound healing and immune suppression are notorious for their role in the tumor microenvironment, increasing tumor cell motility and promoting evasion of anti-tumor immunity. Therefore, current and future studies aimed at targeting important players of efferocytosis should reveal new and efficacious therapeutic approaches for limiting cancer progression and relapse.

Apoptosis and Clearance of the Secretory Mammary Epithelium

The development of the mammary gland occurs in four distinct phases: embryogenesis, puberty, pregnancy, and a post-lactational phase involving profound levels of cell death and tissue remodeling. This post-lactational phase is termed post-lactational involution. During embryogenesis, a solid epithelial bud is generated in the embryonic ectoderm. As this bud continues to grow in cell number, the epithelial bud invaginates into the underlying mesenchyme forming the nascent mammary epithelium. The mammary epithelium grows as solid epithelial cords, lengthening distally and branching to form the rudimentary epithelial network. At puberty, ductal elongation continues in a proximal-to-distal direction, and side branches appear along the ducts. The side branches also lengthen distally, and continue to branch. This pattern of distal growth and branching fills the mouse mammary fat pad with an extensively branched epithelium by the end of puberty [6]. Similar to what is seen during embryonic mammary development and patterning, the mammary ducts developing during puberty originally appear in solid epithelial cords. Apoptosis canalizes the luminal space within the ducts, allowing a patent conduit for milk to traverse through the breast epithelium [1, 7]. Ultimately, the rodent mammary epithelium is comprised of a continuous, branching network leading from the nipple to primary ducts and smaller ductules that terminate in terminal end buds (TEBs), blunt ends or alveoli. The inner luminal cells are separated from the basement membrane by an outer myoepithelial layer. Myoepithelial cells secrete basement membrane components to which the epithelium attaches, and that physically separates the epithelium from the stromal compartment.

Efferocytosis in the Tumor Microenvironment

Cell death in response to physiological cues is a feature of many normal tissues, and often occurs due to tissue damage or disease. Regardless of the reason, dying cells require their rapid removal from the tissue in order to maintain homeostasis in the tissue, prevent potential cytotoxic immunity against self-antigens, and to promote wound healing. Phagocytic removal of dying cells is referred to as efferocytosis, a process necessary for sterile wound healing. Interestingly, malignant tumor progression has long been compared to wound healing, and recent evidence demonstrates a molecular link between efferocytosis and tumor a ‘wound healing’ phenotype. We discuss the impact of efferocytosis in the normal breast and in breast cancers.

Abstract P4-04-02: Cell death and efferocytosis generate a pro-metastatic landscape during mammary gland involution that increase dissemination of post-partum breast cancers

Although pregnancy at a young age decreases a woman’s lifetime breast cancer risk, the first five years following a pregnancy at any age are associated with increased breast cancer risk, a risk that continues to increase with age. Given societal trends to postpone child-birth later into women’s lives, the incidence of malignant post-partum breast cancer is expected to increase. Currently, breast cancers diagnosed 2-5 years post-partum account for nearly 25% of all pre-menopausal breast cancers. Importantly, breast cancers diagnosed 2-5 years post-partum are diagnosed more frequently as metastatic disease and correlate with decreased disease-free survival (DFS) versus breast cancers occurring in other young pre-menopausal women. In contrast, many studies show that breast cancers diagnosed during pregnancy do not correlate with decreased DFS, although there remains some debate about this conclusion. The observation that post-partum breast cancers correlate with reduced DFS suggests that events occurring in the post-partum breast augment the malignant severity of tumors existing therein. This hypothesis is supported by studies in which breast cancer cells transplanted into involuting mouse mammary fatpads grow and invade more rapidly than cells transplanted into mammary glands of nulliparous mice, demonstrating a unique mammary micro-environment during involution versus other reproductive stages. We aimed to study how the changing landscape of the post-partum breast accelerates cancer progression. Although the molecular mechanisms underlying the malignant severity of post-partum breast cancers (ppBCs) are unclear, they relate to stromal wound healing events during post-partum involution, a dynamic process characterized by widespread cell death in milk-producing mammary epithelial cells (MECs). Using both spontaneous and allografted mammary tumors in fully immune-competent mice, we discovered that post-partum involution increased mammary tumor metastasis 10-fold. Widespread cell death occurred not only in MECs, but also in tumor epithelium. Dying tumor cells were cleared through MerTK-dependent efferocytosis, which robustly induced transcription of wound healing cytokines interleukins (IL)-4, -10, and -13, and transforming growth factor (TGF)-beta. Genetic models of MerTK ablation and pharmacologic MerTK inhibition impaired efferocytosis in post-partum tumors, reduced M2-like macrophages without altering total macrophage levels, decreased TGF-beta expression and reduced post-partum tumor metastasis to levels seen in nulliparous mice. TGF-beta blockade reduced post-partum tumor metastasis. These data suggest that widespread cell death during post-partum involution triggers efferocytosis-induced wound healing cytokines in the tumor microenvironment that promote metastatic tumor progression. Citation Format: Rebecca S Cook, Shelton Earp, Jamie Stanford. Cell death and efferocytosis generate a pro-metastatic landscape during mammary gland involution that increase dissemination of post-partum breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-02.