Cx26 drives self-renewal in triple-negative breast cancer via interaction with NANOG and focal adhesion kinase.

Tumors adapt their phenotypes during growth and in response to therapies through dynamic changes in cellular processes. Connexin proteins enable such dynamic changes during development, and their dysregulation leads to disease states. The gap junction communication channels formed by connexins have been reported to exhibit tumor-suppressive functions, including in triple-negative breast cancer (TNBC). However, we find that connexin 26 (Cx26) is elevated in self-renewing cancer stem cells (CSCs) and is necessary and sufficient for their maintenance. Cx26 promotes CSC self-renewal by forming a signaling complex with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK), resulting in NANOG stabilization and FAK activation. This FAK/NANOG-containing complex is not formed in mammary epithelial or luminal breast cancer cells. These findings challenge the paradigm that connexins are tumor suppressors in TNBC and reveal a unique function for Cx26 in regulating the core self-renewal signaling that controls CSC maintenance.

New Advances and Challenges of Targeting Cancer Stem Cells.

The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20-23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222-7. ©2017 AACR.

CD55 regulates self-renewal and cisplatin resistance in endometrioid tumors.

Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

STAT3 activation by leptin receptor is essential for TNBC stem cell maintenance.

Leptin (LEP) binds to the long form of the leptin receptor (LEPRb), leading to the activation of multiple signaling pathways that are potential targets for disrupting the obesity-breast cancer link. In triple-negative breast cancer (TNBC), LEP is hypothesized to predominantly mediate its tumorigenic effects via a subpopulation of LEPRb-positive tumor cells termed cancer stem cells (CSCs) that can initiate tumors and induce tumor progression. Previously, we showed that LEP promotes CSC survival in vivo Moreover, silencing LEPRb in TNBC cells compromised the CSC state. The mechanisms by which LEPRb regulates TNBC CSC intracellular signaling are not clear. We hypothesized that activation of LEPRb signaling is sufficient to drive CSC maintenance in TNBC. Here, we show that activation of LEPRb in non-CSCs isolated using our CSC reporter system resulted in a transition to the stem cell state. In CSCs, LEP induced STAT3 phosphorylation, whereas LEP did not induce STAT3 phosphorylation in non-CSCs. Introduction of constitutively active STAT3 into LEPRb-transfected non-CSCs significantly induced NANOG, SOX2 and OCT4 expression compared with control non-CSCs. To determine the intracellular phospho-tyrosine residue of LEPRb that is necessary for the induction of the stem cell state in non-CSCs, we transfected the tyrosine residue point mutants L985, F1077 and S1138 into non-CSCs. Non-CSCs transfected with the L985 mutant exhibited increased STAT3 phosphorylation, increased SOCS3 expression and an induction of GFP expression compared with non-CSCs expressing the F1077 and S1138 mutants. Our data demonstrate that LEPRb-induced STAT3 activation is essential for the induction and maintenance of TNBC CSCs.

Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression.

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Five-Part Pentameric Nanocomplex Shows Improved Efficacy of Doxorubicin in CD44+ Cancer Cells.

The CD44 receptor is common among many cancer types where overexpression is synonymous with poor prognosis in prostate, glioma, and breast cancer. More notably CD44 overexpression has been shown in a number of different cancer stem cells (CSC) which are present in many solid tumors and drive growth, recurrence, and resistance to conventional therapies. Triple negative breast cancer CSCs correlate to worse prognosis and early relapse due to higher drug resistance and increased tumor heterogeneity and thus are prime targets for anticancer therapy. To specifically target cells overexpressing CD44 receptors, including CSCs, we synthesized a pentameric nanocomplex (PNC) containing gold nanoparticles, doxorubicin (Dox) conjugated to thiolated hyaluronic acid via an acid-labile hydrazone bond, and thiolated poly(ethylene glycol) DNA CD44 aptamer. In vitro drug release was highest at 8 h time point at acidic pH (pH 4.7) and in 10 mM glutathione. The PNC is almost an order of magnitude more effective than Dox alone in CD44+ cells versus CD44 low cells. Functionally, the PNC reduced CSC self-renewal. The PNC provides a therapeutic strategy that can improve the efficiency of Dox and decrease nontargeted toxicity thereby prolonging its use to individual patients.

Transcriptomic-metabolomic reprogramming in EGFR-mutant NSCLC early adaptive drug escape linking TGFß2-bioenergetics-mitochondrial priming.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFß2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFß2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.

Increased cancer stem cell invasion is mediated by myosin IIB and nuclear translocation.

Despite many advances in the treatment of breast cancer, it remains one of the leading causes of death among women. One hurdle for effective therapy is the treatment of the highly invasive and tumorigenic subpopulation of tumors called cancer stem cells (CSCs). CSCs, when stimulated with EGF, migrate through a physiological 3D collagen matrix at a higher velocity than non-stem cancer cells (non-SCCs). This increased invasion is due, in part, by an enhanced nuclear translocation ability of CSCs. We observed no difference between CSC and non-SCC in cellular migration rates on a 2D surface. Furthermore, during transwell migration using large diameter transwell pores, both CSC and non-SCC populations migrated with similar efficiency. However, when challenged with more restrictive transwells, CSCs were dramatically more capable of transwell migration. These results implicate nuclear translocation as a major rate limiting factor for CSC dissemination. We further show that non-muscle myosin IIB is critical for this enhanced nuclear translocation and the ability for cancer stem cells to efficiently migrate through restrictive 3D environments. These studies suggest that cytoskeletal elements upregulated in CSCs, such as myosin IIB, may be valuable targets for intervention in cancer stem cell dispersal from tumors.

Induction of HEXIM1 activities by HMBA derivative 4a1: Functional consequences and mechanism.

We have been studying the role of Hexamethylene bisacetamide (HMBA) Induced Protein 1 (HEXIM1) as a tumor suppressor whose expression is decreased in tamoxifen resistant and metastatic breast cancer. HMBA was considered the most potent and specific inducer for HMBA inducible protein 1 (HEXIM1) prior to our studies. Moreover, the ability of HMBA to induce differentiation is advantageous for its therapeutic use when compared to cytotoxic agents. However, HMBA induced HEXIM1 expression required at mM concentrations and induced dose limiting toxicity, thrombocytopenia. Thus we structurally optimized HMBA and identified a more potent inducer of HEXIM1 expression, 4a1. The studies reported herein tested the ability of 4a1 to induce HEXIM1 activities using a combination of biochemical, cell phenotypic, and in vivo assays. 4a1 induced breast cell differentiation, including the stem cell fraction in triple negative breast cancer cells. Clinically relevant HEXIM1 activities that are also induced by 4a1 include enhancement of the inhibitory effects of tamoxifen and inhibition of breast tumor metastasis. We also provide mechanistic basis for the phenotypic effects of 4a1. Our results support the potential of an unsymmetrical HMBA derivative, such as 4a1, as lead compound for further drug development.

Cisplatin induces stemness in ovarian cancer.

The mainstay of treatment for ovarian cancer is platinum-based cytotoxic chemotherapy. However, therapeutic resistance and recurrence is a common eventuality for nearly all ovarian cancer patients, resulting in poor median survival. Recurrence is postulated to be driven by a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). A current limitation in CSC studies is the inability to interrogate their dynamic changes in real time. Here we utilized a GFP reporter driven by the NANOG-promoter to enrich and track ovarian CSCs. Using this approach, we identified a population of cells with CSC properties including enhanced expression of stem cell transcription factors, self-renewal, and tumor initiation. We also observed elevations in CSC properties in cisplatin-resistant ovarian cancer cells as compared to cisplatin-naïve ovarian cancer cells. CD49f, a marker for CSCs in other solid tumors, enriched CSCs in cisplatin-resistant and -naïve cells. NANOG-GFP enriched CSCs (GFP+ cells) were more resistant to cisplatin as compared to GFP-negative cells. Moreover, upon cisplatin treatment, the GFP signal intensity and NANOG expression increased in GFP-negative cells, indicating that cisplatin was able to induce the CSC state. Taken together, we describe a reporter-based strategy that allows for determination of the CSC state in real time and can be used to detect the induction of the CSC state upon cisplatin treatment. As cisplatin may provide an inductive stress for the stem cell state, future efforts should focus on combining cytotoxic chemotherapy with a CSC targeted therapy for greater clinical utility.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.

Vimentin is a target of PKCß phosphorylation in MCP-1-activated primary human monocytes.

OBJECTIVE AND DESIGN: We designed a study to detect downstream phosphorylation targets of PKCß in MCP-1-induced human monocytes. METHODS: Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCß antisense oligodeoxyribonucleotides (AS-ODN) or a PKCß inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCß AS-ODN or the PKCß inhibitor peptide, were sequenced. RESULTS: Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon (32)P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCß AS-ODN and the PKCß inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCß antibody revealed that increased PKCß becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCß expression and activity. CONCLUSIONS: We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCß in MCP-1-treated monocytes and that PKCß phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494-504, 2013). Taken together our findings suggest that inhibition of PKCß regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCß phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.

Vimentin is an endogenous ligand for the pattern recognition receptor Dectin-1.

AIMS: Atherosclerosis is a chronic inflammatory disorder of cholesterol deposition in monocyte-derived macrophages (MDM) within the arterial wall leading to impingement on the lumen of the vessel. In atherosclerotic lesions, MDM are the primary source of NADPH oxidase-derived superoxide anion (O2⁻) inducing low-density lipoprotein (LDL) oxidation leading to their unregulated uptake of oxidized LDL and foam cell formation. We recently discovered that zymosan potently activates monocyte NADPH oxidase via the non-toll pattern recognition receptor (PRR), Dectin-1. Other PRRs bind endogenous human ligands, yet no such ligands have been identified for Dectin-1. Our hypothesis was that inflammation generates endogenous ligands for Dectin-1 that activate O2⁻ production and thereby contributes to atherogenesis. METHODS AND RESULTS: Human: anti-zymosan antibodies were used to identify similar, cross-reactive epitopes in human atherosclerotic tissue extracts. Immunoblot analysis revealed consistent antibody reactive protein bands on one- and two-dimensional gel electrophoreses. Vimentin was identified by mass spectrometry in the immunoreactive bands across different tissue samples. Direct binding of vimentin to Dectin-1 was observed using BIACORE. Further data revealed that vimentin induces O2⁻ production by human monocytes. Analysis of human atherosclerotic lesions revealed that vimentin was detected extracellularly in the necrotic core and in areas of active inflammation. Vimentin also co-localized with Dectin-1 in macrophage-rich regions where O2⁻ is produced. CONCLUSION: We conclude that vimentin is an endogenous, activating ligand for Dectin-1. Its presence in areas of artery wall inflammation and O2⁻ production suggests that vimentin activates Dectin-1 and contributes to the oxidation of lipids and cholesterol accumulation in atherosclerosis.

MET signaling: novel targeted inhibition and its clinical development in lung cancer.

MET is a versatile receptor tyrosine kinase within the human kinome which is activated by its specific natural ligand hepatocyte growth factor (HGF). MET signaling plays an important physiologic role in embryogenesis and early development, whereas its deregulation from an otherwise quiescent signaling state in mature adult tissues can lead to upregulated cell proliferation, survival, scattering, motility and migration, angiogenesis, invasion, and metastasis in tumorigenesis and tumor progression. Studies have shown that MET pathway is activated in many solid and hematological malignancies, including lung cancer, and can be altered through ligand or receptor overexpression, genomic amplification, MET mutations, and alternative splicing. The MET signaling pathway is known to be an important novel target for therapeutic intervention in human cancer. A number of novel therapeutic agents that target the MET/HGF pathway have been tested in early-phase clinical studies with promising results. Phase 3 studies of MET targeting agents have just been initiated. We will review the MET signaling pathway and biology in lung cancer and the recent clinical development and advances of MET/HGF targeting agents with emphasis on discussion of issues and strategies needed to optimize the personalized therapy and further clinical development.

Protein kinase Cδ is a critical component of Dectin-1 signaling in primary human monocytes.

Zymosan, a mimic of fungal pathogens, and its opsonized form (ZOP) are potent stimulators of monocyte NADPH oxidase, resulting in the production of O(2)(.-), which is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled O(2)(.-) production may contribute to chronic inflammation and tissue injury. Our laboratory has focused on characterizing the signal transduction pathways that regulate NADPH oxidase activity in primary human monocytes. In this study, we examined the involvement of various pattern recognition receptors and found that Dectin-1 is the primary receptor for zymosan stimulation of O(2)(.-) via NADPH oxidase in human monocytes, whereas Dectin-1 and CR3 mediate the activation by ZOP. Further studies identified Syk and Src as important signaling components downstream of Dectin-1 and additionally identified PKCδ as a novel downstream signaling component for zymosan-induced O(2)(.-) as well as phagocytosis. Our results show that Syk and Src association with Dectin-1 is dependent on PKCδ activity and expression and demonstrate direct binding between Dectin-1 and PKCδ. Finally, our data show that PKCδ and Syk but not Src are required for Dectin-1-mediated phagocytosis. Taken together, our data identify Dectin-1 as the major PRR for zymosan in primary human monocytes and identify PKCδ as a novel downstream signaling kinase for Dectin-1-mediated regulation of monocyte NADPH oxidase and zymosan phagocytosis.