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SH2 containing protein tyrosine phosphatase-2 (SHP2) is recognized as a druggable oncogenic phosphatase that is expressed in both tumor cells and immune cells. How tumor cell–autonomous SHP2 contributes to an immunosuppressive tumor microenvironment (TME) and therapeutic failure of immune checkpoint blockades in metastatic breast cancer (MBC) is not fully understood. Herein, we utilized systemic SHP2 inhibition and inducible genetic depletion of SHP2 to investigate immune reprogramming during SHP2 targeting. Pharmacologic inhibition of SHP2 sensitized MBC cells growing in the lung to α-programmed death ligand 1 (α-PD-L1) antibody treatment via relieving T-cell exhaustion induced by checkpoint blockade. Tumor cell–specific depletion of SHP2 similarly reduced pulmonary metastasis and also relieved exhaustion markers on CD8+ and CD4+ cells. Both systemic SHP2 inhibition and tumor cell–autonomous SHP2 depletion reduced tumor-infiltrated CD4+ T cells and M2-polarized tumor-associated macrophages. Analysis of TCGA datasets revealed that phosphorylation of SHP2 is important for immune-cell infiltration, T-cell activation and antigen presentation. To investigate this mechanistically, we conducted in vitro T-cell killing assays, which demonstrated that pretreatment of tumor cells with FGF2 and PDGF reduced the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factor receptor signaling and three-dimensional culture conditions transcriptionally induced PD-L1 via SHP2. Finally, SHP2 inhibition reduced MAPK signaling and enhanced STAT1 signaling, preventing growth factor–mediated suppression of MHC class I. Overall, our findings support the conclusion that tumor cell–autonomous SHP2 is a key signaling node utilized by MBC cells to engage immune-suppressive mechanisms in response to diverse signaling inputs from TME. Significance: Findings present inhibition of SHP2 as a therapeutic option to limit breast cancer metastasis by promoting antitumor immunity.

Corresponding representative dot plots for the quantification in figure 2D-F and additional myeloid composition analysis in the study of D2.A1 model.

SH2 containing protein tyrosine phosphatase-2 (SHP2) is recognized as a druggable oncogenic phosphatase that is expressed in both tumor cells and immune cells. How tumor cell–autonomous SHP2 contributes to an immunosuppressive tumor microenvironment (TME) and therapeutic failure of immune checkpoint blockades in metastatic breast cancer (MBC) is not fully understood. Herein, we utilized systemic SHP2 inhibition and inducible genetic depletion of SHP2 to investigate immune reprogramming during SHP2 targeting. Pharmacologic inhibition of SHP2 sensitized MBC cells growing in the lung to α-programmed death ligand 1 (α-PD-L1) antibody treatment via relieving T-cell exhaustion induced by checkpoint blockade. Tumor cell–specific depletion of SHP2 similarly reduced pulmonary metastasis and also relieved exhaustion markers on CD8+ and CD4+ cells. Both systemic SHP2 inhibition and tumor cell–autonomous SHP2 depletion reduced tumor-infiltrated CD4+ T cells and M2-polarized tumor-associated macrophages. Analysis of TCGA datasets revealed that phosphorylation of SHP2 is important for immune-cell infiltration, T-cell activation and antigen presentation. To investigate this mechanistically, we conducted in vitro T-cell killing assays, which demonstrated that pretreatment of tumor cells with FGF2 and PDGF reduced the cytotoxicity of CD8+ T cells in a SHP2-dependent manner. Both growth factor receptor signaling and three-dimensional culture conditions transcriptionally induced PD-L1 via SHP2. Finally, SHP2 inhibition reduced MAPK signaling and enhanced STAT1 signaling, preventing growth factor–mediated suppression of MHC class I. Overall, our findings support the conclusion that tumor cell–autonomous SHP2 is a key signaling node utilized by MBC cells to engage immune-suppressive mechanisms in response to diverse signaling inputs from TME.Significance:Findings present inhibition of SHP2 as a therapeutic option to limit breast cancer metastasis by promoting antitumor immunity.

This is the code information for publicly available data analysis.

These are supplementary tables to provide detailed information about the materials used in the study including cell, culture conditions, growth factors, inhibitors, and antibodies.

Ca1a cells (labeled by d-tomato) can migrate off of the solid support scaffold on FN-expressing Ca1h cells.

Control Ca1h cells forming fibrillar strains when cultured on our 3D culture scaffold.

Supplementary Data from Preclinical Development of the Class-I–Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors

Ca1a cells (labeled by d-tomato) can migrate and proliferate on FN-fibrils.

Therapeutic targeting of late-stage breast cancer is limited by an inadequate understanding of how tumor cell signaling evolves during metastatic progression and by the currently available small molecule inhibitors capable of targeting these processes. Herein, we demonstrate that both β3 integrin and fibroblast growth factor receptor-1 (FGFR1) are part of an epithelial–mesenchymal transition (EMT) program that is required to facilitate metastatic outgrowth in response to fibroblast growth factor-2 (FGF2). Mechanistically, β3 integrin physically disrupts an interaction between FGFR1 and E-cadherin, leading to a dramatic redistribution of FGFR1 subcellular localization, enhanced FGF2 signaling and increased three-dimensional (3D) outgrowth of metastatic breast cancer cells. This ability of β3 integrin to drive FGFR signaling requires the enzymatic activity of focal adhesion kinase (FAK). Consistent with these mechanistic data, we demonstrate that FGFR, β3 integrin, and FAK constitute a molecular signature capable of predicting decreased survival of patients with the basal-like subtype of breast cancer. Importantly, covalent targeting of a conserved cysteine in the P-loop of FGFR1–4 with our newly developed small molecule, FIIN-4, more effectively blocks 3D metastatic outgrowth as compared with currently available FGFR inhibitors. In vivo application of FIIN-4 potently inhibited the growth of metastatic, patient-derived breast cancer xenografts and murine-derived metastases growing within the pulmonary microenvironment. Overall, the current studies demonstrate that FGFR1 works in concert with other EMT effector molecules to drive aberrant downstream signaling, and that these events can be effectively targeted using our novel therapeutics for the treatment of the most aggressive forms of breast cancer. Mol Cancer Ther; 15(9); 2096–106. ©2016 AACR.

This file includes supplementary figures S1-S8 and supplementary tables S1-S3.

Ca1h cells depleted for FN (shFN30) fail to form any fibrillar strains when cultured on our 3D culture scaffold.

FN-depleted Ca1h cells (shFN30) cannot facilitate the migration of Ca1a cells (labeled in red) off of the solid support of the scaffold.

S1. Differential fluorescent labeling of epithelial and mesenchymal cells. S2. Ca1h cells are not metastatic. S3. Fibronectin coated polystyrene induces a partial epithelialmesenchymal transition. S4. Fibronectin is in a fibular form in patient tumors. S5. Fibrillar fibronectin induction of EMT is dependent on the expression of b1 integrin. S6. Fibrillar fibronectin induces a mesenchymal phenotype. S7. Depletion of b1 increases intracellular FN. S8. Intracellular FN colocalizes with the actin cytoskeleton. S9. Systemically dormant cells express intracellular FN. S10.Depletion of FN increases pulmonary tumor proliferation.

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I–selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

Ca1a cells (labeled by d-tomato) can migrate and proliferate on FN-fibrils.

FN-depleted Ca1h cells (shFN32) cannot facilitate the migration of Ca1a cells (labeled in red) off of the solid support of the scaffold.

Supplementary Figure from Preclinical Development of the Class-I–Selective Histone Deacetylase Inhibitor OKI-179 for the Treatment of Solid Tumors

The table lists the phosphorylation values for the array of peptides containing tyrosine or serine/threonine phosphorylation sites. This array was used to compare lysates from HME2 parental cells, TGF-beta-induced EMT and lapatinib-induced EMT.

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here, we compare a reversible model of epithelial–mesenchymal transition (EMT) induced by TGFβ to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGFβ-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal–epithelial transition (MET). Genetic knockout of autophagy-related 7 (ATG7) or pharmacologic inhibition of SYK activity with fostamatinib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, this study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells.Significance:These findings present inhibition of spleen tyrosine kinase as a therapeutic option to limit breast cancer metastasis by promoting systemic tumor dormancy.See related commentary by Farrington and Narla, p. 1756

This file contain supplemental figures S1 through S4. These figures present additional immunoblot and replicate immune fluorescent frames to further support the conclusions made from the data presented in the main manuscript.

Histone deacetylases (HDACs) play critical roles in epigenomic regulation, and histone acetylation is dysregulated in many human cancers. Although HDAC inhibitors are active in T-cell lymphomas, poor isoform selectivity, narrow therapeutic indices, and a deficiency of reliable biomarkers may contribute to the lack of efficacy in solid tumors. In this article, we report the discovery and preclinical development of the novel, orally bioavailable, class-I–selective HDAC inhibitor, OKI-179. OKI-179 and its cell active predecessor OKI-005 are thioester prodrugs of the active metabolite OKI-006, a unique congener of the natural product HDAC inhibitor largazole. OKI-006, OKI-005, and subsequently OKI-179, were developed through a lead candidate optimization program designed to enhance physiochemical properties without eroding potency and selectivity relative to largazole. OKI-005 displays antiproliferative activity in vitro with induction of apoptosis and increased histone acetylation, consistent with target engagement. OKI-179 showed antitumor activity in preclinical cancer models with a favorable pharmacokinetic profile and on-target pharmacodynamic effects. Based on its potency, desirable class I HDAC inhibition profile, oral bioavailability, and efficacy against a broad range of solid tumors, OKI-179 is currently being evaluated in a first-in-human phase I clinical trial with plans for continued clinical development in solid tumor and hematologic malignancies.

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wntlung) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions.

Metabolic reprogramming and immune evasion are established hallmarks of the tumor microenvironment (TME). Tumor metabolic dysregulation is a key mediator of tumor immune evasion. High levels of lactate produced by the tumor potently suppress antitumor immunity in the TME. Pyruvate carboxylase (PC), the enzyme responsible for the anaplerotic conversion of pyruvate to oxaloacetate, is required for lung metastasis in triple negative breast cancer (TNBC). Moreover, PC may be dispensable in some cells within the TME, and loss of PC expression is associated with immunosuppression. Here we test whether PC suppression in two murine models of TNBC alters tumor metabolism and promotes immunosuppression. C57BL6/J mice were orthotopically injected with E0771 or M-Wnt tumor cells bearing shRNA either targeting PC or a scramble control (n=3-6/group for each experiment). Tumor growth was monitored by digital calipers. RNA isolated from tumors was used for transcriptomic profiling using Affymetrix Clariom D microarray. Gene set enrichment analysis was used to identify pathways and processes enriched in each condition, as well as to define a hypoxia signature associated with PC expression. Immunohistochemistry was performed using an anti-CD3 antibody to identify T cell infiltration. Untargeted metabolomics, extracellular flux analysis and high resolution respirometry were used to assay metabolic effects of PC suppression. We found that suppression of PC in multiple animal models promotes primary tumor growth (~2 fold increase in size) and alters metabolic, transcriptomic and immunohistochemical markers of tumor immunosurveillance. Specifically, suppression of PC drove reduction of OCR, which was restored when exogenous malate was provided to ensure anaplerosis was non-limiting. Simultaneously, lactate production was elevated by PC suppression, as was in vitro sensitivity to inhibition of lactate metabolism. Critically, we identified that loss of immunosurrveilance following PC suppression could be restored by inhibition of lactate transport. Finally, we demonstrated that PC expression is markedly lower in tumors of patients whose transcriptomic profile indicated higher levels of hypoxia, and confirmed in vitro that hypoxia can directly suppress PC. Taken together, these data demonstrate hypoxia-driven PC suppression may be a key mechanism through which primary tumors limit antitumor immunity in part via enhanced lactate production. Thus, these data highlight that PC-directed tumor metabolism is a nexus of tumor progression and antitumor immunity. Ongoing work will examine the immune profile of the TME from PC suppressed tumors, and identify if PC overexpression is sufficient to enhance antitumor immunity. Citation Format: Michael F. Coleman, Eylem Kulkoyluoglu Cotul, Alexander J. Pfeil, Emily N. Devericks, Muhammad H. Safdar, Hao Chen, Violet A. Kiesel, Dorothy Teegarden, Stephen D. Hursting, Michael K. Wendt. Suppression of pyruvate carboxylase drives tumor microenvironment immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 656.

Metastatic breast cancer (MBC) is the most advanced stage of breast cancer. Our understanding of the molecular mechanisms which drive MBC remain incomplete. Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) promote drug resistance and metastasis. It has been reported that fibroblast growth factor receptor 1 (FGFR1) plays a key role during the EMT:MET cycle. Furthermore, FGFR1 is amplified in 13% of primary and 20% of metastatic breast cancer patients. Therefore, optimizing inhibition of FGFR1 is crucial for the therapeutic targeting of the late stage breast cancer. First, we examined the efficacies of FGFR kinase inhibitors in the murine based dormant 4T07 tumor model. Inhibition of FGFR kinase activity leads to tumor growth inhibition but fail to eradicate dormant breast cancer cells. Therefore, we explored broader approaches to inhibit FGFR1 expression in addition to blockade of its kinase activity. G-quadruplex (G4) structures are secondary DNA structures commonly found upstream of transcriptional start sites (TSS) of oncogenes restricting their expression. Consequently, pharmacological stabilization of G4 structures within the promoters of cancer-related genes via use of small molecules has emerged as a promising therapeutic approach in cancer. Results herein demonstrate that the proximal promoter of FGFR1 contains sequences that form G4. Circular dichroism was used to verify formation of G4 in the FGFR1 proximal promoter. Importantly, use of the G4-binding compound CX-5461 stabilized the FGFR1 G4 structure, blocked the transcriptional activity of the FGFR1 proximal promoter and decreased FGFR1 expression. Therefore, we implemented the G4 stabilizers in FGFR1 expressing and metastatic drug-resistant cell lines. This approach results in dramatic downregulation of FGFR1 in the protein level after treatment with the G4 stabilizer. G4 stabilizing agents also interfere with constitutive FGFR1 and EMT-driven FGFR1 expression. Importantly, use of the G4-targeting compound CX5461 effectively blocked FGFR1 expression and inhibited FGFR1 downstream signaling, resulting in eradication of dormant breast cancer cells using a 3D culture model system. Finally, in vivo application of CX5461 reduced pulmonary tumor growth and prolonged animal survival in an FGFR1-driven model of metastasis. In conclusion, our evaluation of FGFR kinase inhibitors validates clinically observed resistance to this approach in MBC. Moreover, our findings suggest that G4 stabilization may be a potential therapeutic strategy for FGFR1 expressing MBC. Citation Format: Muhammad Hassan Safdar, Hang Lin, Sarah Dagher, Jonathan Dickerhoff, Danzhou Yang, Michael K. Wendt. Targeted down regulation of FGFR1 through G-quadruplex stabilization in metastatic breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5005.

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that efficiently delivers a highly potent microtubule inhibitor to HER2 overexpressing cells. Herein, we utilize HER2 transformed human mammary epithelial cells (HME2) to demonstrate in vitro and in vivo response and recurrence upon T-DM1 treatment. Continuous in vitro dosing of HME2 cells with T-DM1 failed to produce a spontaneously resistant cell line. However, induction of epithelial–mesenchymal transition (EMT) via pretreatment with TGF-β1 was capable of promoting emergence of T-DM1-resistant (TDM1R) cells. Flow cytometric analyses indicated that induction of EMT decreased trastuzumab binding, prior to overt loss of HER2 expression in TDM1R cells. Kinome analyses of TDM1R cells indicated increased phosphorylation of ErbB1, ErbB4, and FGFR1. TDM1R cells failed to respond to the ErbB kinase inhibitors lapatinib and afatinib, but they acquired sensitivity to FIIN4, a covalent FGFR kinase inhibitor. In vivo, minimal residual disease (MRD) remained detectable via bioluminescent imaging following T-DM1-induced tumor regression. Upon cessation of the ADC, relapse occurred and secondary tumors were resistant to additional rounds of T-DM1. These recurrent tumors could be inhibited by FIIN4. Moreover, ectopic overexpression of FGFR1 was sufficient to enhance tumor growth, diminish trastuzumab binding, and promote recurrence following T-DM1-induced MRD. Finally, patient-derived xenografts from a HER2+ breast cancer patient who had progressed on trastuzumab failed to respond to T-DM1, but tumor growth was significantly inhibited by FIIN4. Overall, our studies strongly support therapeutic combination of TDM1 with FGFR-targeted agents in HER2+ breast cancer.

The effectiveness of immunotherapy as a treatment for metastatic breast cancer is limited due to low numbers of infiltrating lymphocytes in metastatic lesions. Herein, we demonstrated that adjuvant therapy using FIIN4, a covalent inhibitor of fibroblast growth factor receptor (FGFR), dramatically delayed the growth of pulmonary metastases in syngeneic models of metastatic breast cancer. In addition, we demonstrated in a syngeneic model of systemic tumor dormancy that targeting of FGFR enhanced the immunogenicity of the pulmonary tumor microenvironment through increased infiltration of CD8+ lymphocytes and reduced presence of myeloid suppressor cells. Similar impacts on immune cell infiltration were observed upon genetic depletion of FGFR1 in tumor cells, which suggested a direct influence of FGFR signaling on lymphocyte trafficking. Suppression of CD8+ lymphocyte infiltration was consistent with FGFR-mediated inhibition of the T-cell chemoattractant CXCL16. Initial attempts to concomitantly administer FIIN4 with immune checkpoint blockade failed due to inhibition of immune-mediated tumor cell killing via blockade of T-cell receptor signaling by FIIN4. However, this was overcome by using a sequential dosing protocol that consisted of FIIN4 treatment followed by anti–PD-L1. These data illustrate the complexities of combining kinase inhibitors with immunotherapy and provide support for further assessment of FGFR targeting as an approach to enhance antitumor immunity and improve immunotherapy response rates in patients with metastatic breast cancer.

Metabolic reprogramming and immune evasion are established hallmarks of the tumor microenvironment (TME). Growing evidence supports tumor metabolic dysregulation as an important mediator of tumor immune evasion. High TME levels of lactate potently suppress antitumor immunity. Pyruvate carboxylase (PC), the enzyme responsible for the anaplerotic conversion of pyruvate to oxaloacetate, is essential for lung metastasis in breast cancer. Moreover, PC may be dispensable in some cells within the TME, and loss of PC expression is associated with immunosuppression. Here we test whether PC suppression alters tumor metabolism and immunosuppression. Using multiple animal models of breast cancer, we identify a dimorphic role for PC expression in mammary cancer cells. Specifically, PC supports metastatic colonization of the lungs, while suppression of PC promotes primary tumor growth and suppresses histological and transcriptomic markers of antitumor immunity. We demonstrate that PC is potently suppressed by hypoxia, and that PC is frequently suppressed in solid tumors, particularly those with higher levels of hypoxia. Using metabolomics, high-resolution respirometry, and extracellular flux analysis, we show that PC-suppressed cells produce more lactate and undergo less oxidative phosphorylation than controls. Finally, we identify lactate metabolism as a targetable dependency of PC-suppressed cells, which is sufficient to restore T cell populations to the TME of PC-suppressed tumors. Taken together, these data demonstrate that elevated lactate following PC suppression by hypoxia may be a key mechanism through which primary tumors limit antitumor immunity. Thus, these data highlight that PC-directed tumor metabolism is a nexus of tumor progression and antitumor immunity.

Acquisition of resistance to targeted therapies remains a major clinical obstacle for the HER2+ subtype of breast cancer. Using an isogeneic progression series of HER2+ breast cancer metastasis we demonstrate that metastatic cells have an increased capacity to acquire resistance to the covalent, pan-ErbB inhibitor, neratinib. RNA sequencing analyses comparing parental and metastatic cells identified upregulation of transglutaminase 2 (TG2). Genetic depletion and overexpression approaches established that TG2 is both necessary and sufficient for acquisition of neratinib resistance. Mechanistically, we describe a pathway in which TG2-mediates activation of NF-κB signaling leading to upregulation of IL-6 in metastatic cells. This autocrine expression of IL-6 functions to maintain enhanced levels of TG2 via JAK:STAT3 signaling. This drug persistence feedback loop can be interrupted through the use of the JAK1/2 inhibitor ruxolitinib. In vivo application of ruxolitinib had no effect on tumor growth under non-treated conditions, but effectively prevented acquisition of resistance, leading to tumor regression upon coadministration with neratinib. Overall, our studies reveal a mechanism in metastatic breast cancer cells that predisposes them to acquisition of resistance to ErbB-targeted therapeutics. Clinically, immediate application of ruxolitinib could prevent acquisition of resistance and improve patient responses to HER2-targeted therapies.

Human epidermal growth factor receptor 2 (HER2)-amplified breast cancers are treated using targeted antibodies and kinase inhibitors, but resistance to these therapies leads to systemic tumor recurrence of metastatic disease. Herein, we conducted gene expression analyses of HER2 kinase inhibitor-resistant cell lines as compared to their drug-sensitive counterparts. These data demonstrate the induction of epithelial–mesenchymal transition (EMT), which included enhanced expression of fibroblast growth factor receptor 1 (FGFR1) and axonal guidance molecules known as neuropilins (NRPs). Immunoprecipitation of FGFR1 coupled with mass spectroscopy indicated that FGFR1 forms a physical complex with NRPs, which is enhanced upon induction of EMT. Confocal imaging revealed that FGFR1 and NRP1 predominantly interact throughout the cytoplasm. Along these lines, short hairpin RNA-mediated depletion of NRP1, but not the use of NRP1-blocking antibodies, inhibited FGFR signaling and reduced tumor cell growth in vitro and in vivo. Our results further indicate that NRP1 upregulation during EMT is mediated via binding of the chromatin reader protein, bromodomain containing 4 (BRD4) in the NRP1 proximal promoter region. Pharmacological inhibition of BRD4 decreased NRP1 expression and ablated FGF-mediated tumor cell growth. Overall, our studies indicate that NRPs facilitate aberrant growth factor signaling during EMT-associated drug resistance and metastasis. Pharmacological combination of epigenetic modulators with FGFR-targeted kinase inhibitors may provide improved outcomes for breast cancer patients with drug-resistant metastatic disease.

Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of bypassing current targeted therapies via engagement of several growth promoting pathways. SH2 containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase known to facilitate growth and survival signaling downstream of numerous receptor inputs. Herein, we used inducible genetic depletion and two distinct pharmacological inhibitors to investigate the therapeutic potential of targeting SHP2 in MBC. Cells that acquired resistance to the ErbB kinase inhibitor, neratinib, displayed increased phosphorylation of SHP2 at the Y542 activation site. In addition, higher levels of SHP2 phosphorylation, but not expression, were associated with decreased survival of breast cancer patients. Pharmacological inhibition of SHP2 activity blocked ERK1/2 and AKT signaling generated from exogenous stimulation with FGF2, PDGF, and hGF and readily prevented MBC cell growth induced by these factors. SHP2 was also phosphorylated upon engagement of the extracellular matrix (ECM) via focal adhesion kinase. Consistent with the potential of SHP2-targeted compounds as therapeutic agents, the growth inhibitory property of SHP2 blockade was enhanced in ECM-rich 3D culture environments. In vivo blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis and extended the survival of systemic tumor-bearing mice. Finally, inhibition of SHP2 in combination with FGFR-targeted kinase inhibitors synergistically blocked the growth of MBC cells. Overall, our findings support the conclusion that SHP2 constitutes a shared signaling node allowing MBC cells to simultaneously engage a diversity of growth and survival pathways, including those derived from the ECM.

Several cancers, including breast cancers, show dependence on glutamine metabolism. The purpose of the present study was to determine the mechanistic basis and impact of differential glutamine metabolism in nonmetastatic and metastatic murine mammary cancer cells. Universally labeled 13C5-glutamine metabolic tracing, qRT-PCR, measures of reductive–oxidative balance, and exogenous ammonium chloride treatment were used to assess glutamine reprogramming. Results show that 4 mM media concentration of glutamine, compared with 2 mM, reduced viability only in metastatic cells, and that this decrease in viability was accompanied by increased incorporation of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle. While increased glutamine metabolism in metastatic cells occurred in tandem with a decrease in the reduced/oxidized glutathione ratio, treatment with the antioxidant molecule N-acetylcysteine did not rescue cell viability. However, the viability of metastatic cells was more sensitive to ammonium chloride treatment compared with nonmetastatic cells, suggesting a role of metabolic reprogramming in averting nitrogen cytotoxicity in nonmetastatic cells. Overall, these results demonstrate the ability of nonmetastatic cancer cells to reprogram glutamine metabolism and that this ability may be lost in metastatic cells.

Recent progress with pulsar timing array experiments, especially from the NANOGrav collaboration indicate that we are on the cusp of detecting significant signals from the inspiral of super-massive black hole binaries (SMBHB). While recent analysis has focused on nearby galaxies as possible sources of the loudest signals, we show that mergers in identified clusters at z~1 can have larger strain amplitudes. We make an estimate comparing the nearby 2MASS redshift survey galaxy sample with the more distant MaDCoWS cluster sample, showing that the latter might be expected to contribute more, and louder, gravitational wave events. Thus the first individual source detections may well be from ultra-massive BH in clusters at z~1, rather than nearby galaxies., 7 pages, to Appear in the Astrophysical Journal

Coronavirus disease 2019 (COVID-19) is an emerging human infectious disease caused by severe acute respiratory syndrome 2 (SARS-CoV-2, initially called novel coronavirus 2019-nCoV) virus. Thus, an accurate and specific diagnosis of COVID-19 is urgently needed for effective point-of-care detection and disease management. The reported promise of two-dimensional (2D) transition-metal carbides (Ti3C2Tx MXene) for biosensing owing to a very high surface area, high electrical conductivity, and hydrophilicity informed their selection for inclusion in functional electrodes for SARS-CoV-2 detection. Here, we demonstrate a new and facile functionalization strategy for Ti3C2Tx with probe DNA molecules through noncovalent adsorption, which eliminates expensive labeling steps and achieves sequence-specific recognition. The 2D Ti3C2Tx functionalized with complementary DNA probes shows a sensitive and selective detection of nucleocapsid (N) gene from SARS-CoV-2 through nucleic acid hybridization and chemoresistive transduction. The fabricated sensors are able to detect the SARS-CoV-2 N gene with sensitive and rapid response, a detection limit below 105 copies/mL in saliva, and high specificity when tested against SARS-CoV-1 and MERS. We hypothesize that the MXenes’ interlayer spacing can serve as molecular sieving channels for hosting organic molecules and ions, which is a key advantage to their use in biomolecular sensing.

Accurately replicating and analyzing cellular responses to mechanical cues is vital for exploring metastatic disease progression. However, many of the existing in vitro platforms for applying mechanical stimulation seed cells on synthetic substrates. To better recapitulate physiological conditions, a novel actuating platform is developed with the ability to apply tensile strain on cells at various amplitudes and frequencies in a high-throughput multi-well culture plate using a physiologically-relevant substrate. Suspending fibrillar fibronectin across the body of the magnetic actuator provides a matrix representative of early metastasis for 3D cell culture that is not reliant on a synthetic substrate. This platform enables the culturing and analysis of various cell types in an environment that mimics the dynamic stretching of lung tissue during normal respiration. Metabolic activity, YAP activation, and morphology of breast cancer cells are analyzed within one week of cyclic stretching or static culture. Further, matrix degradation is significantly reduced in breast cancer cell lines with metastatic potential after actuation. These new findings demonstrate a clear suppressive cellular response due to cyclic stretching that has implications for a mechanical role in the dormancy and reactivation of disseminated breast cancer cells to macrometastases.

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]

Background: Throughout metastatic progression, cancer cells acquire anchorage independence, or the ability to survive detached from the extracellular matrix (ECM). While untransformed epithelial cells reduce energy metabolism when detached, cancer cells display metabolic flexibility to continue important metabolic processes. Glucose and glutamine are predominant nutrients utilized for energy as well as other purposes, and their metabolism is regulated by cancer cells.Methods: The purpose of the current studies was to determine the effects of detachment on glucose and glutamine metabolism in human breast epithelial MCF10A cells transfected with the Harvey-ras oncogene (MCF10A-ras), a model of early-stage cancer. Detachment was simulated with poly-HEMA coated plates, and intracellular metabolic flux was determined using stably labeled 13C5-glutamine and 13C6-glucose tracers.Results: Results show reduced glutamine flux in detached cells as determined by reduced accumulation of label in glutamate (21%), malate (30%), and aspartate (23%) from 13C5-glutamine. Detachment also reduced flux of 13C6-glucose to pyruvate and lactate pools by 51% and 29%, respectively. Similarly, detachment reduced total intracellular pool sizes of pyruvate (51%), lactate (49%), α-ketoglutarate (43%), fumarate (32%), malate (19%), alanine (35%), serine (35%), and glutamate (28%) compared to attached cells, but citrate and aspartate pool sizes were unchanged. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 131% and 190%, respectively. In detachment, PC activity, determined by 13C6-glucose derived M + 3 isotopomers, was shown to preferentially replenish malate and aspartate, but not citrate pools. In addition, doxycycline-inducible shRNA depletion of PC significantly decreased, while doxycycline-inducible PC overexpression significantly increased, detached cell viability. Further, a switch from glutamine to PC activity for anaplerosis was demonstrated, as supplementation with the cell permeable analog of the tricarboxylic acid cycle intermediate, α-ketoglutarate, a downstream metabolite of glutamine, decreased PC mRNA abundance in detached cells.Conclusion: Collectively, these results suggest that detached breast cancer cells increase PC activity in response to decreased glutamine-derived anaplerosis to promote cell survival.

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.