qRT-PCR. Whole-tumor RNA was harvested with an RNeasy kit (QIAGEN), and cDNA was synthesized (High Capacity; Applied Biosystems) and amplified using the murine cDNA-specific primers (Integrated DNA Technologies) listed in Supplemental Methods, along with SYBR Green Supermix (Bio-Rad). The following primers were used: MRC1 (forward: 5′ CCCTCAGCAAGCGATGTGC 3′; reverse: 5′-GGATACTTGCCAGGT CCCCA-3′); iNOS (forward: 5′-GGAGCATCCCAAGTACGAGTGG-3′; reverse: 5′-CGGCC-CACTTCCTCCAG); IL10 (forward: 5′-GGCGCTGTCATC-GATTTCTCC; reverse: 5′-GGCCTTGTAGACACCTTGGTC); Tgfb1 (forward: 5′-CGCAACAACGCCATCTATGAG; reverse: 5′-CGG-GACAGCAATGGGGGTTC); IL4 (forward: 5′-GGTCACAGGAGAAGG-GACG; reverse: 5′-GCGAAGCACCTTGGAAGCC);, IL12b (forward: 5′-GGAGTGGGATGTGTCCTCAG; reverse: 5′-CGGGAGTCCAGTC-CACCTCT); CCL3 (forward: 5′-CCACTGCCCTTGCTGTTCTTCTCT; reverse: 5′-GGGTGTCAGCTCCATATGGCG); and Rplp0 (forward: 5′-TCCTATAAAAGGCACACGCGGGC; reverse: 5′-AGACGATGT-CACTCCAACGAGGACG). Target To generate apoptotic MCF7, cells were treated in suspension with 1 μm BKM120 plus 2 μm ABT-263 (both inhibitors from Selleck Chemicals) for 4 hours, washed 5 times with PBS to remove residual drug, and used directly for efferocytosis assays or for annexin V staining. For efferocytosis coculture assays, Raw264.7-GFP cells (10 4 /well) and PyVmT or MCF7 cells (72 hours after infection with Ad.mCherry and Ad.HS-V-TK) were seeded together in a monolayer in 24-well plates in 2% FBS and cultured for 24 hours prior to the addition PBS or gancyclovir. Cells were imaged at 8, 16, and 32 h after addition of gancyclovir. Cells were collected and counted under fluorescence after 32 hours of coculture. In some experiments, Raw264.7-GFP cells (10 4 / well) were seeded in a monolayer in 24-well plates and cultured for 24 hours prior to the addition of 10 3 live MCF7-mCherry or 10 3 dead MCF7-mCherry cells in serum-free media. Where indicated in the figures, BMS-777607 (1 μm) or a neutralizing goat anti-mouse MerTK antibody (AF591, 25 μg/ml; R&D Systems)(44) was added 2 hours prior to the addition of gancyclovir or 2 hours prior to the addition of dead MCF7 cells to macrophage monolayers. Live and dead MCF7 cells were similarly seeded without Raw264.7 cells as single cultures. Media were collected after 16 hours of coculture, passed through a 0.2-μm filter, and used neat (250 μl) to quantify murine IL-10 and IL-4 by ELISA (BioLegend) according to the manufacturer's protocol. Total remaining cells were collected after 16 hours of coculture, lysed, and RNA was collected using an RNeasy kit (QIAGEN). MethodsMice. All mice were inbred on an FVB background for more than 10 generations. WT FVB, MMTV PyVmT and MerTK -/-mice (67), originally referred to as Mer KD , were purchased from The Jackson Laboratory. Mice were genotyped by PCR of genomic DNA as previously described(30). Female virgin mice were randomized into 2 groups: (a) 1 group that remained virgin, and (b) 1 group that was bred from 42 to 44 days of age with WT male mice. Pregnancies were timed according to identification of a va...

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.

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 paracrinesignaling mechanisms.A berrant regulation of the ErbB family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers (1-4). This family consists of four members: HER1/ErbB1/ EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Except for ErbB3, which has weak kinase activity, the ErbB RTKs exhibit dimerization-induced phosphorylation and catalytic activation. In response to ligand binding, ErbBs form homodimers and heterodimers with other ErbB coreceptors. ErbB3 relies on transphosphorylation by heterodimeric partners to induce signal transduction (5-7).ErbB RTKs are required for breast development, although each receptor bears a unique spatiotemporal expression pattern. ErbB2 loss in the mammary epithelium delays ductal elongation during puberty and disorganizes cells within terminal end buds (TEBs) (8-10). EGFR and ErbB4 are not required for mammary ductal development. Rather, EGFR is expressed in the basal epithelium (BE) and in the mammary stroma, and ErbB4 is necessary for milk production (11,12). Although classical knockout of mouse ErbB3 results in embryonic lethality (13), transplant experiments showed that ErbB3 drives growth of the mammary epithelium during puberty (8). Although the mechanism(s) by which ErbB2 and ErbB3 regulate growth of the ductal epithelium are currently unknown, such knowledge will impact our understanding of the earliest events contributing to the formation of ErbB2/HER2-amplified breast cancers, which account for 20-30% of all breast cancers. ErbB3-ErbB2 heterodimers are the most potent oncogenic ErbB-signaling pair due in part to strong ErbB3-induced phosphatidylinositol 3-kinase (PI3K) activation in response to ErbB3 tyrosine phosphorylation at six PI3K interaction motifs (14,15).To understand the role of ErbB3 in mammary gland dev...

IntroductionAberrant regulation of the erythroblastosis oncogene B (ErbB) family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers (1-4). This family consists of 4 related members, HER1/ErbB1/EGFR, HER2/ErbB2/Neu, HER3/ ErbB3, and HER4/ErbB4. Except for ErbB3, which has very weak kinase activity, the ErbB RTKs exhibit dimerization-induced tyrosine phosphorylation and catalytic activation that results in signal transduction to intracellular targets. ErbBs are able to form homodimers as well as heterodimers with other coreceptors of the ErbB family. ErbB3 relies on transphosphorylation by heterodimeric partners to induce signal transduction (5-7). Therefore, therapeutic interest in the ErbB family has been historically focused on EGFR and ErbB2.HER2/ErbB2 is gene amplified in nearly 25% of all breast cancers. Targeting HER2/ErbB2 activity using the monoclonal antibody trastuzumab or the small molecule tyrosine kinase inhibitor (TKI) lapatinib decreases growth of HER2-amplified breast cancer cells, improving survival and outcome of patients with breast cancers.Recently, clinical results demonstrate that therapeutic resistance to HER2/ErbB2 inhibitors occurs in part due to feedback upregulation of ErbB3 signaling, increasing the activity and output through the PI3K/mTOR pathway. This observation may underlie the recently

Mortality from pancreatic ductal adenocarcinoma cancer (PDAC) is among the highest of any cancer and frontline therapy has changed little in years. Activation of endothelial nitric oxide synthase (eNOS or NOS III) has been implicated recently in the pathogenesis of PDAC. In this study, we used genetically engineered mouse and human xenograft models to evaluate the consequences of targeting eNOS in PDAC. Genetic deficiency in eNOS limited the development of pre-invasive pancreatic lesions and trended towards an extended lifespan in mice with advanced pancreatic cancer. These effects were also observed upon oral administration of the clinically evaluated NOS small molecule inhibitor L-NAME. Similarly, other transgenic models of oncogenic KRas-driven tumors responded to L-NAME treatment. Finally, these results were recapitulated in xenograft models of human pancreatic cancer, in which L-NAME was found to broadly inhibit tumorigenic growth. Taken together, our findings offer preclinical proof-of-principle to repurpose L-NAME for clinical investigations in treatment of PDAC and possibly other KRas-driven human cancers.

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.

Mutations in

ErbB3, a member of the ErbB family of receptor tyrosine kinases, is a potent activator of phosphatidyl inositol-3 kinase (PI3K) and mTOR signaling, driving tumor cell survival and therapeutic resistance in breast cancers. In luminal breast cancers, ErbB3 upregulation following treatment with the anti-estrogen 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 datasets. LRIG1, an estrogen-inducible ErbB down-regulator, 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 down-regulate ErbB3. Importantly, LRIG1 overexpression improved fulvestrant-mediated growth inhibition, while cells expressing the LRIG1 mutant were poorly sensitive to fulvestrant, despite effective ERα down-regulation. Consistent with these results, LRIG1 expression correlated positively with increased disease-free survival in anti-estrogen-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.