Meghan M. Morrison

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.

The receptor tyrosine kinase ErbB3 maintains the balance between luminal and basal breast epithelium

ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations. ErbB3 loss in mammary epithelial cells shifted gene expression patterns toward a mammary basal cell/stem cell signature. ErbB3 depletion-induced gene expression changes were rescued upon activation of Akt and MAPK signaling. Interestingly, proliferation and expansion of the mammary basal epithelium (BE) occurred upon ErbB3 targeting in the luminal epithelium, but not upon its targeting in the BE. Multiple cytokines, including interleukin 6, were induced upon ErbB3 depletion in luminal epithelium cells, which increased growth of BE cells. Taken together, these results suggest that ErbB3 regulates the balance of differentiated breast epithelial cell types by regulating their growth and survival through autocrine- and paracrine-signaling mechanisms.

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.

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.

mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Decreased LRIG1 in fulvestrant-treated luminal breast cancer cells permits ErbB3 upregulation and increased growth

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mammalian target of rapamycin (mTOR) signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the antiestrogen fulvestrant enhances PI3K/mTOR-mediated cell survival. However, the mechanism by which ErbB3 is upregulated in fulvestrant-treated cells is unknown. We found that ErbB3 protein levels and cell surface presentation were increased following fulvestrant treatment, focusing our attention on proteins that regulate ErbB3 at the cell surface, including Nrdp1, NEDD4 and LRIG1. Among these, only LRIG1 correlated positively with ERα, but inversely with ErbB3 in clinical breast cancer data sets. LRIG1, an estrogen-inducible ErbB downregulator, was decreased in a panel of fulvestrant-treated luminal breast cancer cells. Ectopic LRIG1 expression from an estrogen-independent promoter uncoupled LRIG1 from estrogen regulation, thus sustaining LRIG1 and maintaining low ErbB3 levels in fulvestrant-treated cells. An LRIG1 mutant lacking the ErbB3 interaction motif was insufficient to downregulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, whereas cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα downregulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in antiestrogen-treated breast cancer patients. These data suggest that ERα-dependent expression of LRIG1 dampens ErbB3 signaling in luminal breast cancer cells, and by blocking ERα activity with fulvestrant, LRIG1 is decreased thus permitting ErbB3 accumulation, enhanced ErbB3 signaling to cell survival pathways and blunting therapeutic response to fulvestrant.

Abstract 5: Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers

We found that 8.1% of luminal breast cancer cases curated by The Cancer Genome Atlas (TCGA), harbor gene amplification of anti-apoptotic Bcl-2 family members, warranting further study of anti-apoptotic Bcl-2 family proteins (A1, Bcl-2, Bcl-xL, Bcl-w and Mcl-1) in luminal breast cancers. A complex balance of pro- and anti-apoptotic Bcl-2 proteins guides activation versus repression of the intrinsic apoptotic pathway, with overexpression of anti-apoptotic factors tipping the balance towards cell survival. This mechanism is commonly used by cancers to evade apoptosis. We used the BH3-mimetic ABT-263 to inhibit Bcl-2, Bcl-xL and Bcl-w in a panel of human luminal breast cancer lines, causing only modest apoptosis in monolayer and growth inhibition in three-dimensional (3D) matrix. Expression and activity of Mcl-1, another anti-apoptotic Bcl-2 family member, was rapidly upregulated in response to ABT-263 through increased cap-dependent MCL1 translation. Mcl-1 knock-down decreased tumor cell growth and improved ABT-263-mediated tumor cell killing. Inhibition of cap-dependent translation using the mTOR inhibitor RAD001/everolimus or the EIF4 complex inhibitor 4E1RCat blocked Mcl-1 induction in ABT-263 treated cells, enhancing apoptosis and growth inhibition. In vivo, single agent ABT-263 was ineffective at reducing tumor growth in xenografted human breast cancers and in transgenic WAP-Myc luminal mammary tumors. However, RAD001 decreased Mcl-1 levels and decreased WAP-Myc tumor growth by 40%. These results demonstrate that heightened cap-dependent translation of Mcl-1 prevents therapeutically induced tumor cell killing in ABT-263 treated luminal tumors, highlighting the importance of Mcl-1 regulation in therapeutic resistance, and identifying Mcl-1 as a potential therapeutic target in luminal breast cancers. These studies suggest that targeted inhibition of Mcl-1 alone or in combination with other Bcl-2 family inhibitors will enhance tumor cell killing in luminal breast cancers. Citation Format: Michelle M. Williams, Linus Lee, Violeta Sanchez, Meghan M. Morrison, Donna Hicks, Rebecca S. Cook. Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5. doi:10.1158/1538-7445.AM2015-5

Abstract B07: Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers

In the context of cancer, the intrinsic apoptotic pathway is exploited to favor tumor cell survival through overexpression of anti-apoptotic Bcl-2 family members (Bcl-A1, Bcl-2, Bcl-xL, Bcl-w and Mcl-1). We investigated targeting of anti-apoptotic Bcl-2 proteins in a panel of human luminal breast cancer cell lines. Use of ABT-263, the Bcl-2/Bcl-xL/Bcl-w inhibitor, induced transient tumor cell killing and decreased tumor cell growth in only 1 of 4 cell lines in three dimensional (3D) cultures. Mcl-1 expression and activity were rapidly upregulated upon ABT-263 treatment, highlighting the compensatory nature of Mcl-1. In luminal breast cancers, Mcl-1 was the most frequently amplified anti-apoptotic Bcl-2 family member according to The Cancer Genome Atlas, while nearly 80% of the luminal breast tumor epithelium was positive for Mcl-1 in a tissue microarray. Thus, we hypothesized that Mcl-1 may be a dominant tumor cell survival factor in luminal breast cancers. Use of the mTOR inhibitor RAD001 (everolimus) to target Mcl-1 indirectly decreased tumor cell growth and increased tumor cell killing in 3 of 4 cell lines, as well as WAP-Myc luminal mammary tumors, demonstrating the effectiveness of Mcl-1 as a therapeutic target in breast cancers. While Mcl-1 inhibition alone did not affect growth of T47D cells, robust growth inhibition and tumor cell killing were seen in all cell lines upon inhibition of Mcl-1 using RAD001 or a EIF4 complex inhibitor (4E1RCat) in combination with ABT-263, suggesting that the induction of Mcl-1 upon ABT-263 treatment may be supported by increased cap-dependent translation. These results demonstrate that the sensitivity of luminal breast cancers to ABT-263 is enhanced by Mcl-1 inhibition, warranting further investigation into inhibition of anti-apoptotic Bcl-2 family proteins, in particular Mcl-1, as a clinical strategy to improve survival of patients with luminal breast cancers. Citation Format: Michelle M. Williams, Linus Lee, Meghan M. Morrison, Courtney McKernan, Violeta Sanchez, Donna Hicks, Thomas Stricker, Rebecca S. Cook. Mcl-1-mediated resistance to ABT-263 is combated by mTOR inhibition in luminal breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B07.

Abstract B09: Targeting the anti-apoptotic protein Mcl-1 with the mTOR inhibitor RAD001 sensitizes luminal breast cancers to the Bcl-2/Bcl-xL inhibitor ABT-263

In an effort to enhance therapeutic tumor cell killing in luminal breast cancers, we investigated the inhibition of the anti-apoptotic Bcl-2 family proteins Bcl-2, Bcl-xL and Mcl-1. Anti-apoptotic Bcl-2 proteins are inhibitors of the intrinsic apoptotic pathway that sequester and inhibit pro-apoptotic Bcl-2 family members. Sustained overexpression of anti-apoptotic Bcl-2 family members frequently occurs in cancers and supports tumor initiation, tumor progression, therapeutic resistance and poor patient survival. While only 2/324 and 1/324 luminal breast cancers curated by The Cancer Genome Atlas (TCGA) had gene amplification of BCL2 and BCL2L1 (encoding Bcl-xL) respectively, 21/324 ( MCL1 amplification. Wap-Myc transgenic mouse mammary tumors, which are highly enriched for the luminal A transcription signature, displayed profoundly increased Mcl-1 levels as compared to normal mammary tissue, suggesting a role for Mcl-1 in luminal breast cancer biology. Using a panel of four MCL1 -amplified breast cancer cell lines, we found that two were marginally sensitive to treatment with ABT-263, a BH3-mimetic that targets Bcl-2 and Bcl-xL. Conversely, ectopic overexpression of Mcl-1 decreased sensitivity to ABT-263. ABT-263 induced minimal cell killing in MCF7 xenografts and had no effect on tumor growth. Similarly, ABT-263 did not affect tumor growth in luminal-like Wap-Myc mouse mammary tumors. Thus, luminal breast cancer cells expressing increased Mcl-1 are poorly responsive to Bcl-2/Bcl-xL inhibition. Upregulation of Mcl-1 expression and activity, an established ABT-263-resistance mechanism in leukemias and lymphomas, was observed in MCL1 -amplified luminal breast cancer cell lines treated with ABT-263 in culture and in vivo. ABT-263 also increased the anti-apoptotic activity of Mcl-1 towards the pro-apoptotic Bcl-2 family member BIM, suggesting that Mcl-1 upregulation compensates for inhibition of Bcl-2 and Bcl-xL by ABT-263. There are currently no Mcl-1 specific inhibitors. However, Mcl-1 undergoes cap-dependent translation, and thus inhibition of cap-dependent translation has been proposed as a method to inhibit the protein expression of Mcl-1. Importantly, the mTOR pathway is required for efficient cap-dependent translation in many cancer cells. We found that the rapalogue RAD001/everolimus, an mTOR inhibitor, decreased Mcl-1 protein levels in MCL1 -amplified luminal breast cancer cell lines. Furthermore, RAD001 blocked Mcl-1 protein upregulation in response to ABT-263, producing increased cell killing and decreased cell growth in all MCL1 -amplified luminal breast cancer cell lines. Treatment of luminal Wap-Myc mammary tumors with RAD001 decreased tumor volume by 40%, while treatment with ABT-263 had no effect on tumor volume. These data suggest that Mcl-1 targeting may be superior to Bcl-2/Bcl-xL targeting in luminal breast cancers, particularly in luminal breast cancers with MCL1 -amplification. Furthermore, we have identified the mTOR pathway as a potential therapeutic target for blocking Mcl-1 expression in luminal breast cancers. Citation Format: Michelle M. Williams, Linus Lee, Meghan M. Morrison, Andrew J. Williams, Violeta Sanchez, Donna Hicks, Rebecca Cook. Targeting the anti-apoptotic protein Mcl-1 with the mTOR inhibitor RAD001 sensitizes luminal breast cancers to the Bcl-2/Bcl-xL inhibitor ABT-263. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B09.