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.

ErbB3 drives mammary epithelial survival and differentiation during pregnancy and lactation.

BACKGROUND: During pregnancy, as the mammary gland prepares for synthesis and delivery of milk to newborns, a luminal mammary epithelial cell (MEC) subpopulation proliferates rapidly in response to systemic hormonal cues that activate STAT5A. While the receptor tyrosine kinase ErbB4 is required for STAT5A activation in MECs during pregnancy, it is unclear how ErbB3, a heterodimeric partner of ErbB4 and activator of phosphatidyl inositol-3 kinase (PI3K) signaling, contributes to lactogenic expansion of the mammary gland. METHODS: We assessed mRNA expression levels by expression microarray of mouse mammary glands harvested throughout pregnancy and lactation. To study the role of ErbB3 in mammary gland lactogenesis, we used transgenic mice expressing WAP-driven Cre recombinase to generate a mouse model in which conditional ErbB3 ablation occurred specifically in alveolar mammary epithelial cells (aMECs). RESULTS: Profiling of RNA from mouse MECs isolated throughout pregnancy revealed robust Erbb3 induction during mid-to-late pregnancy, a time point when aMECs proliferate rapidly and undergo differentiation to support milk production. Litters nursed by ErbB3 KO dams weighed significantly less when compared to litters nursed by ErbB3 WT dams. Further analysis revealed substantially reduced epithelial content, decreased aMEC proliferation, and increased aMEC cell death during late pregnancy. Consistent with the potent ability of ErbB3 to activate cell survival through the PI3K/Akt pathway, we found impaired Akt phosphorylation in ErbB3 KO samples, as well as impaired expression of STAT5A, a master regulator of lactogenesis. Constitutively active Akt rescued cell survival in ErbB3-depleted aMECs, but failed to restore STAT5A expression or activity. Interestingly, defects in growth and survival of ErbB3 KO aMECs as well as Akt phosphorylation, STAT5A activity, and expression of milk-encoding genes observed in ErbB3 KO MECs progressively improved between late pregnancy and lactation day 5. We found a compensatory upregulation of ErbB4 activity in ErbB3 KO mammary glands. Enforced ErbB4 expression alleviated the consequences of ErbB3 ablation in aMECs, while combined ablation of both ErbB3 and ErbB4 exaggerated the phenotype. CONCLUSIONS: These studies demonstrate that ErbB3, like ErbB4, enhances lactogenic expansion and differentiation of the mammary gland during pregnancy, through activation of Akt and STAT5A, two targets crucial for lactation.

Two distinct mTORC2-dependent pathways converge on Rac1 to drive breast cancer metastasis.

BACKGROUND: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. METHODS: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. RESULTS: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility. CONCLUSION: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.

Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study.

Although new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2-prostanoid receptor (TPr), a G-protein-coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for CPI211, we performed a phenome-wide association study (PheWAS) of the gene encoding TPr, TBXA2R, using robust deidentified health records and matched genomic data from more than 29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single-nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated 200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered an rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced transendothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as antimetastatic agents and highlighting the use of PheWAS as an approach to accelerate drug repurposing.

Therapeutic inhibition of Mcl-1 blocks cell survival in estrogen receptor-positive breast cancers.

Cancers often overexpress anti-apoptotic Bcl-2 proteins for cell death evasion, a recognized hallmark of cancer progression. While estrogen receptor (ER)-α+ breast cancers express high levels of three anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1), pharmacological inhibition of Bcl-2 and/or Bcl-xL fails to induce cell death in ERα+ breast cancer cell lines, due to rapid and robust Mcl-1 upregulation. The mechanisms of acute Mcl-1 upregulation in response to Bcl-2/Bcl-xL inhibition remain undefined in in ERα+ breast cancers. We report here that blockade of Bcl-2 or Bcl-xL, alone or together, rapidly induced mTOR signaling in ERα+ breast cancer cells, rapidly increasing cap-dependent Mcl-1 translation. Cells treated with a pharmacological inhibitor of cap-dependent translation, or with the mTORC1 inhibitor RAD001/everolimus, displayed reduced protein levels of Mcl-1 under basal conditions, and failed to upregulate Mcl-1 protein expression following treatment with ABT-263, a pharmacological inhibitor of Bcl-2 and Bcl-xL. Although treatment with ABT-263 alone did not sustain apoptosis in tumor cells in culture or in vivo, ABT-263 plus RAD001 increased apoptosis to a greater extent than either agent used alone. Similarly, combined use of the selective Mcl-1 inhibitor VU661013 with ABT-263 resulted in tumor cell apoptosis and diminished tumor growth in vivo. These findings suggest that rapid Mcl-1 translation drives ABT-263 resistance, but can be combated directly using emerging Mcl-1 inhibitors, or indirectly through existing and approved mTOR inhibitors.

Treatment-Induced Tumor Cell Apoptosis and Secondary Necrosis Drive Tumor Progression in the Residual Tumor Microenvironment through MerTK and IDO1.

Efferocytosis is the process by which apoptotic cells are cleared from tissue by phagocytic cells. The removal of apoptotic cells prevents them from undergoing secondary necrosis and releasing their inflammation-inducing intracellular contents. Efferocytosis also limits tissue damage by increasing immunosuppressive cytokines and leukocytes and maintains tissue homeostasis by promoting tolerance to antigens derived from apoptotic cells. Thus, tumor cell efferocytosis following cytotoxic cancer treatment could impart tolerance to tumor cells evading treatment-induced apoptosis with deleterious consequences in tumor residual disease. We report here that efferocytosis cleared apoptotic tumor cells in residual disease of lapatinib-treated HER2+ mammary tumors in MMTV-Neu mice, increased immunosuppressive cytokines, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). Blockade of efferocytosis induced secondary necrosis of apoptotic cells, but failed to prevent increased tumor MDSCs, Treg, and immunosuppressive cytokines. We found that efferocytosis stimulated expression of IFN-γ, which stimulated the expression of indoleamine-2,3-dioxegenase (IDO) 1, an immune regulator known for driving maternal-fetal antigen tolerance. Combined inhibition of efferocytosis and IDO1 in tumor residual disease decreased apoptotic cell- and necrotic cell-induced immunosuppressive phenotypes, blocked tumor metastasis, and caused tumor regression in 60% of MMTV-Neu mice. This suggests that apoptotic and necrotic tumor cells, via efferocytosis and IDO1, respectively, promote tumor 'homeostasis' and progression. SIGNIFICANCE: These findings show in a model of HER2+ breast cancer that necrosis secondary to impaired efferocytosis activates IDO1 to drive immunosuppression and tumor progression.

Staphylococcus xylosus Cystitis and Struvite Urolithiasis in Nude Mice Implanted with Sustained-release Estrogen Pellets.

Female nude mice (J:NU-Foxn1nu; age, 6 wk) were injected with 1 million MCF7 human breast cancer cells in the fourth mammary fat pads and received a 21-d sustained-release estrogen pellet (0.25 mg) subcutaneously in the dorsum of the neck. All mice were maintained in sterile housing and provided sterile water and irradiated rodent chow. Approximately 6 wk after implantation, 4 of the 30 mice showed clinical signs of depression and dehydration. The 2 animals most severely affected were euthanized and presented for necropsy. The urinary bladders of these animals were distended with variable sized white, opaque uroliths. Urinalysis revealed coccal bacteria, erythrocytes, neutrophils and struvite crystals. Urine cultures from both necropsied animals grew heavy, pure growths of Staphylococcus xylosus. The organism was sensitive to all antibiotics tested except erythromycin (intermediate). Analysis of the uroliths revealed 100% struvite composition. Remaining mice in the study were evaluated clinically for hydration status, the ability to urinate, and the presence of palpable stones in the urinary bladder; one additional mouse had a firm, nonpainful bladder (urolithiasis suspected). Given the sensitivity of the organisms cultured from urine samples, the remaining mice were placed on enrofloxacin in the drinking water (0.5 mg/mL). All remaining mice completed the study without further morbidity or mortality. Previous studies have reported the association of estrogen supplementation with urinary bladder pathology, including infection and urolithiasis. Here we present a case of urolithiasis and cystitis in nude mice receiving estrogen supplementation that was associated with Staphylococcus xylosus, which previously was unreported in this context. When assessing these nude mice for urolithiasis, we found that visualizing the stones through the body wall, bladder palpation, and bladder expression were helpful in identifying affected mice.

Therapeutically Active RIG-I Agonist Induces Immunogenic Tumor Cell Killing in Breast Cancers.

Cancer immunotherapies that remove checkpoint restraints on adaptive immunity are gaining clinical momentum but have not achieved widespread success in breast cancers, a tumor type considered poorly immunogenic and which harbors a decreased presence of tumor-infiltrating lymphocytes. Approaches that activate innate immunity in breast cancer cells and the tumor microenvironment are of increasing interest, based on their ability to induce immunogenic tumor cell death, type I IFNs, and lymphocyte-recruiting chemokines. In agreement with reports in other cancers, we observe loss, downregulation, or mutation of the innate viral nucleotide sensor retinoic acid-inducible gene I (RIG-I/DDX58) in only 1% of clinical breast cancers, suggesting potentially widespread applicability for therapeutic RIG-I agonists that activate innate immunity. This was tested using an engineered RIG-I agonist in a breast cancer cell panel representing each of three major clinical breast cancer subtypes. Treatment with RIG-I agonist resulted in upregulation and mitochondrial localization of RIG-I and activation of proinflammatory transcription factors STAT1 and NF-κB. RIG-I agonist triggered the extrinsic apoptosis pathway and pyroptosis, a highly immunogenic form of cell death in breast cancer cells. RIG-I agonist also induced expression of lymphocyte-recruiting chemokines and type I IFN, confirming that cell death and cytokine modulation occur in a tumor cell-intrinsic manner. Importantly, RIG-I activation in breast tumors increased tumor lymphocytes and decreased tumor growth and metastasis. Overall, these findings demonstrate successful therapeutic delivery of a synthetic RIG-I agonist to induce tumor cell killing and to modulate the tumor microenvironment in vivo Significance: These findings describe the first in vivo delivery of RIG-I mimetics to tumors, demonstrating a potent immunogenic and therapeutic effect in the context of otherwise poorly immunogenic breast cancers. Cancer Res; 78(21); 6183-95. ©2018 AACR.

Intrinsic apoptotic pathway activation increases response to anti-estrogens in luminal breast cancers.

Estrogen receptor-α positive (ERα+) breast cancer accounts for approximately 70-80% of the nearly 25,0000 new cases of breast cancer diagnosed in the US each year. Endocrine-targeted therapies (those that block ERα activity) serve as the first line of treatment in most cases. Despite the proven benefit of endocrine therapies, however, ERα+ breast tumors can develop resistance to endocrine therapy, causing disease progression or relapse, particularly in the metastatic setting. Anti-apoptotic Bcl-2 family proteins enhance breast tumor cell survival, often promoting resistance to targeted therapies, including endocrine therapies. Herein, we investigated whether blockade of anti-apoptotic Bcl-2 family proteins could sensitize luminal breast cancers to anti-estrogen treatment. We used long-term estrogen deprivation (LTED) of human ERα+ breast cancer cell lines, an established model of sustained treatment with and acquired resistance to aromatase inhibitors (AIs), in combination with Bcl-2/Bcl-xL inhibition (ABT-263), finding that ABT-263 induced only limited tumor cell killing in LTED-selected cells in culture and in vivo. Interestingly, expression and activity of the Bcl-2-related factor Mcl-1 was increased in LTED cells. Genetic Mcl-1 ablation induced apoptosis in LTED-selected cells, and potently increased their sensitivity to ABT-263. Increased expression and activity of Mcl-1 was similarly seen in clinical breast tumor specimens treated with AI + the selective estrogen receptor downregulator fulvestrant. Delivery of Mcl-1 siRNA loaded into polymeric nanoparticles (MCL1 si-NPs) decreased Mcl-1 expression in LTED-selected and fulvestrant-treated cells, increasing tumor cell death and blocking tumor cell growth. These findings suggest that Mcl-1 upregulation in response to anti-estrogen treatment enhances tumor cell survival, decreasing response to therapeutic treatments. Therefore, strategies blocking Mcl-1 expression or activity used in combination with endocrine therapies would enhance tumor cell death.

Selective mTORC2 Inhibitor Therapeutically Blocks Breast Cancer Cell Growth and Survival.

Small-molecule inhibitors of the mTORC2 kinase (torkinibs) have shown efficacy in early clinical trials. However, the torkinibs under study also inhibit the other mTOR-containing complex mTORC1. While mTORC1/mTORC2 combined inhibition may be beneficial in cancer cells, recent reports describe compensatory cell survival upon mTORC1 inhibition due to loss of negative feedback on PI3K, increased autophagy, and increased macropinocytosis. Genetic models suggest that selective mTORC2 inhibition would be effective in breast cancers, but the lack of selective small-molecule inhibitors of mTORC2 have precluded testing of this hypothesis to date. Here we report the engineering of a nanoparticle-based RNAi therapeutic that can effectively silence the mTORC2 obligate cofactor Rictor. Nanoparticle-based Rictor ablation in HER2-amplified breast tumors was achieved following intratumoral and intravenous delivery, decreasing Akt phosphorylation and increasing tumor cell killing. Selective mTORC2 inhibition in vivo, combined with the HER2 inhibitor lapatinib, decreased the growth of HER2-amplified breast cancers to a greater extent than either agent alone, suggesting that mTORC2 promotes lapatinib resistance, but is overcome by mTORC2 inhibition. Importantly, selective mTORC2 inhibition was effective in a triple-negative breast cancer (TNBC) model, decreasing Akt phosphorylation and tumor growth, consistent with our findings that RICTOR mRNA correlates with worse outcome in patients with basal-like TNBC. Together, our results offer preclinical validation of a novel RNAi delivery platform for therapeutic gene ablation in breast cancer, and they show that mTORC2-selective targeting is feasible and efficacious in this disease setting.Significance: This study describes a nanomedicine to effectively inhibit the growth regulatory kinase mTORC2 in a preclinical model of breast cancer, targeting an important pathogenic enzyme in that setting that has been undruggable to date. Cancer Res; 78(7); 1845-58. ©2018 AACR.

Key Survival Factor, Mcl-1, Correlates with Sensitivity to Combined Bcl-2/Bcl-xL Blockade.

An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1-selective inhibitors for targeted tumor cell killing.Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259-68. ©2016 AACR.

Impaired tumor microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression.

EphA2 belongs to a unique family of receptor tyrosine kinases that play critical roles in development and disease. Since EphA2 is required for ephrin-A1 ligand-induced vascular remodeling and is overexpressed in a variety of vascularized human adenocarcinomas, we assessed tumor angiogenesis and metastatic progression in EphA2-deficient host animals. 4T1 metastatic mammary adenocarcinoma cells transplanted subcutaneously and orthotopically into EphA2-deficient female mice displayed decreased tumor volume, tumor cell survival, microvascular density, and lung metastasis relative to tumor-bearing littermate controls. To determine if the phenotype in EphA2-deficient mice was endothelial cell intrinsic, we also analyzed endothelial cells isolated from EphA2-deficient animals for their ability to incorporate into tumor vessels in vivo, as well as to migrate in response to tumor-derived signals in vitro. EphA2-deficient endothelial cells displayed impaired survival and failed to incorporate into tumor microvessels in vivo, and displayed impaired tumor-mediated migration in vitro relative to controls. These data suggest that host EphA2 receptor tyrosine kinase function is required in the tumor microenvironment for tumor angiogenesis and metastatic progression.

Rictor/mTORC2 Drives Progression and Therapeutic Resistance of HER2-Amplified Breast Cancers.

HER2 overexpression drives Akt signaling and cell survival and HER2-enriched breast tumors have a poor outcome when Akt is upregulated. Akt is activated by phosphorylation at T308 via PI3K and S473 via mTORC2. The importance of PI3K-activated Akt signaling is well documented in HER2-amplified breast cancer models, but the significance of mTORC2-activated Akt signaling in this setting remains uncertain. We report here that the mTORC2 obligate cofactor Rictor is enriched in HER2-amplified samples, correlating with increased phosphorylation at S473 on Akt. In invasive breast cancer specimens, Rictor expression was upregulated significantly compared with nonmalignant tissues. In a HER2/Neu mouse model of breast cancer, genetic ablation of Rictor decreased cell survival and phosphorylation at S473 on Akt, delaying tumor latency, penetrance, and burden. In HER2-amplified cells, exposure to an mTORC1/2 dual kinase inhibitor decreased Akt-dependent cell survival, including in cells resistant to lapatinib, where cytotoxicity could be restored. We replicated these findings by silencing Rictor in breast cancer cell lines, but not silencing the mTORC1 cofactor Raptor (RPTOR). Taken together, our findings establish that Rictor/mTORC2 signaling drives Akt-dependent tumor progression in HER2-amplified breast cancers, rationalizing clinical investigation of dual mTORC1/2 kinase inhibitors and developing mTORC2-specific inhibitors for use in this setting. Cancer Res; 76(16); 4752-64. ©2016 AACR.

Optical metabolic imaging identifies glycolytic levels, subtypes, and early-treatment response in breast cancer.

Abnormal cellular metabolism is a hallmark of cancer, yet there is an absence of quantitative methods to dynamically image this powerful cellular function. Optical metabolic imaging (OMI) is a noninvasive, high-resolution, quantitative tool for monitoring cellular metabolism. OMI probes the fluorescence intensities and lifetimes of the autofluorescent metabolic coenzymes reduced NADH and flavin adenine dinucleotide. We confirm that OMI correlates with cellular glycolytic levels across a panel of human breast cell lines using standard assays of cellular rates of glucose uptake and lactate secretion (P < 0.05, r = 0.89). In addition, OMI resolves differences in the basal metabolic activity of untransformed from malignant breast cells (P < 0.05) and between breast cancer subtypes (P < 0.05), defined by estrogen receptor and/or HER2 expression or absence. In vivo OMI is sensitive to metabolic changes induced by inhibition of HER2 with the antibody trastuzumab (herceptin) in HER2-overexpressing human breast cancer xenografts in mice. This response was confirmed with tumor growth curves and stains for Ki67 and cleaved caspase-3. OMI resolved trastuzumab-induced changes in cellular metabolism in vivo as early as 48 hours posttreatment (P < 0.05), whereas fluorodeoxyglucose-positron emission tomography did not resolve any changes with trastuzumab up to 12 days posttreatment (P > 0.05). In addition, OMI resolved cellular subpopulations of differing response in vivo that are critical for investigating drug resistance mechanisms. Importantly, OMI endpoints remained unchanged with trastuzumab treatment in trastuzumab-resistant xenografts (P > 0.05). OMI has significant implications for rapid cellular-level assessment of metabolic response to molecular expression and drug action, which would greatly accelerate drug development studies.

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.

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.

Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials.

PURPOSE: Knowledge of tumor mutation status is becoming increasingly important for the treatment of cancer, as mutation-specific inhibitors are being developed for clinical use that target only sub-populations of patients with particular tumor genotypes. Melanoma provides a recent example of this paradigm. We report here development, validation, and implementation of an assay designed to simultaneously detect 43 common somatic point mutations in 6 genes (BRAF, NRAS, KIT, GNAQ, GNA11, and CTNNB1) potentially relevant to existing and emerging targeted therapies specifically in melanoma. METHODS: The test utilizes the SNaPshot method (multiplex PCR, multiplex primer extension, and capillary electrophoresis) and can be performed rapidly with high sensitivity (requiring 5-10% mutant allele frequency) and minimal amounts of DNA (10-20 nanograms). The assay was validated using cell lines, fresh-frozen tissue, and formalin-fixed paraffin embedded tissue. Clinical characteristics and the impact on clinical trial enrollment were then assessed for the first 150 melanoma patients whose tumors were genotyped in the Vanderbilt molecular diagnostics lab. RESULTS: Directing this test to a single disease, 90 of 150 (60%) melanomas from sites throughout the body harbored a mutation tested, including 57, 23, 6, 3, and 2 mutations in BRAF, NRAS, GNAQ, KIT, and CTNNB1, respectively. Among BRAF V600 mutations, 79%, 12%, 5%, and 4% were V600E, V600K, V600R, and V600M, respectively. 23 of 54 (43%) patients with mutation harboring metastatic disease were subsequently enrolled in genotype-driven trials. CONCLUSION: We present development of a simple mutational profiling screen for clinically relevant mutations in melanoma. Adoption of this genetically-informed approach to the treatment of melanoma has already had an impact on clinical trial enrollment and prioritization of therapy for patients with the disease.

BRAF(L597) mutations in melanoma are associated with sensitivity to MEK inhibitors.

UNLABELLED: Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40% to 50% of cases are positive. To uncover other potential targetable mutations, we conducted whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, and D816) wild-type melanoma. Surprisingly, we found a somatic BRAF(L597R) mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF(V600) mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition. A patient with BRAF(L597S) mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data show clinical significance to BRAF(L597) mutations in melanoma. SIGNIFICANCE: This study shows that cells harboring BRAF(L597R) mutants are sensitive to MEK inhibitor treatment, providing a rationale for routine screening and therapy of BRAF(L597R)-mutant melanoma.

Hairpin DNA coated gold nanoparticles as intracellular mRNA probes for the detection of tyrosinase gene expression in melanoma cells.

A new intracellular mRNA imaging probe has been developed that incorporates thiol-terminated hairpin oligonucleotides covalently bound to the surface of citrate-capped gold nanoparticles. The hairpin DNA-coated gold nanoparticles (hAuNPs) positively identifies tyrosinase mRNA in cultured melanoma cells.