Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor-mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid-derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune-suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self-renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5-flurouracil (5-FU) in a low-dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient-derived CSCs but not nonstem tumor cells selectively drove MDSC-mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune-suppressive enzyme arginase-1 in MDSCs in a CXCR2-dependent manner, whereas blocking MIF reduced arginase-1 production. Similarly to 5-FU, targeting tumor-derived MIF conferred a survival advantage to tumor-bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self-renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026-2039.

Inducing Mitotic Catastrophe as a Therapeutic Approach to Improve Outcomes in Ewing Sarcoma.

Ewing sarcoma (EWS) is an aggressive pediatric malignancy of the bone and soft tissues in need of novel therapeutic options. To identify potential therapeutic targets, we focused on essential biological pathways that are upregulated by EWS-FLI1, the primary oncogenic driver of EWS, including mitotic proteins such as Aurora kinase A (AURKA) and kinesin family member 15 (KIF15) and its binding partner, targeting protein for Xklp2 (TPX2). KIF15/TPX2 cooperates with KIF11, a key mitotic kinesin essential for mitotic spindle orientation. Given the lack of clinical-grade KIF15/TPX2 inhibitors, we chose to target KIF11 (using SB-743921) in combination with AURKA (using VIC-1911) given that phosphorylation of KIF15S1169 by Aurora A is required for its targeting to the spindle. In vitro, the drug combination demonstrated strong synergy (Bliss score ≥ 10) at nanomolar doses. Colony formation assay revealed significant reduction in plating efficiency (1-3%) and increased percentage accumulation of cells in the G2/M phase with the combination treatment (45-52%) upon cell cycle analysis, indicating mitotic arrest. In vivo studies in EWS xenograft mouse models showed significant tumor reduction and overall effectiveness: drug combination vs. vehicle control (p ≤ 0.01), SB-743921 (p ≤ 0.01) and VIC-1911 (p ≤ 0.05). Kaplan-Meier curves demonstrated superior overall survival with the combination compared to vehicle or monotherapy arms (p ≤ 0.0001).

Identification of small extracellular vesicle protein biomarkers for pediatric Ewing Sarcoma.

Ewing Sarcoma (EWS) is the second most common osseous malignancy in children and young adults after osteosarcoma, while it is the fifth common osseous malignancy within adult age population. The clinical presentation of EWS is quite often non-specific, with the most common symptoms at presentation consisting of pain, swelling or general discomfort. The dearth of clinically relevant diagnostic or predictive biomarkers continues to remain a pressing clinical challenge. Identification of tumor specific biomarkers can lend towards an early diagnosis, expedited initiation of therapy, monitoring of therapeutic response, and early detection of recurrence of disease. We carried-out a complex analysis of cell lines and cell line derived small extracellular vesicles (sEVs) using label-free-based Quantitative Proteomic Profiling with an intent to determine shared and distinct features of these tumor cells and their respective sEVs. We analyzed EWS cells with different EWS-ETS fusions (EWS-FLI1 type I, II, and III and EWS-ERG) and their corresponding sEVs. Non-EWS controls included osteosarcoma, rhabdomyosarcoma, and benign cells, i.e., osteoid osteoma and mesenchymal stem cells. Proteomic profiling identified new shared markers between cells and their corresponding cell-derived sEVs and markers which were exclusively enriched in EWS-derived sEVs. These exo-biomarkers identified were validated by in silico approaches of publicly available protein databases and by capillary electrophoresis based western analysis (Wes). Here, we identified a protein biomarker named UGT3A2 and found its expression highly specific to EWS cells and their sEVs compared to control samples. Clinical validation of UGT3A2 expression in patient tumor tissues and plasma derived sEV samples demonstrated its specificity to EWS, indicating its potential as a EWS biomarker.

SerpinB3 drives cancer stem cell survival in glioblastoma.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs.

Pretreatment with LCK inhibitors chemosensitizes cisplatin-resistant endometrioid ovarian tumors.

BACKGROUND: Ovarian cancer is the most fatal gynecologic malignancy in the United States. While chemotherapy is effective in the vast majority of ovarian cancer patients, recurrence and resistance to standard systemic therapy is nearly inevitable. We discovered that activation of the non-receptor tyrosine kinase Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK) promoted cisplatin resistance. Here, we hypothesized that treating high grade, platinum resistant endometrioid cancer cells with an LCK inhibitor (LCKi) followed by co-treatment with cisplatin would lead to increased cisplatin efficacy. Our objective was to assess clinical outcomes associated with increased LCK expression, test our hypothesis of utilizing LCKi as pre-treatment followed by co-treatment with cisplatin in platinum resistant ovarian cancer in vitro, and evaluate our findings in vivo to assess LCKi applicability as a therapeutic agent. RESULTS: Kaplan-Meier (KM) plotter data indicated LCK expression is associated with significantly worse median progression-free survival (HR 3.19, p = 0.02), and a trend toward decreased overall survival in endometrioid ovarian tumors with elevated LCK expression (HR 2.45, p = 0.41). In vitro, cisplatin resistant ovarian endometrioid cells treated first with LCKi followed by combination LCKi-cisplatin treatment showed decreased cell viability and increased apoptosis. Immunoblot studies revealed LCKi led to increased expression of phosphorylated H2A histone family X ([Formula: see text]-H2AX), a marker for DNA damage. In vivo results demonstrate treatment with LCKi followed by LCKi-cisplatin led to significantly slowed tumor growth. CONCLUSIONS: We identified a strategy to therapeutically target cisplatin resistant endometrioid ovarian cancer leading to chemosensitization to platinum chemotherapy via treatment with LCKi followed by co-treatment with LCKi-cisplatin.

JAM-A functions as a female microglial tumor suppressor in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary brain tumor and has a dismal prognosis. Previously, we identified that junctional adhesion molecule A (JAM-A), a cell adhesion molecule, is highly elevated in human GBM cancer stem cells and predicts poor patient prognosis. While JAM-A is also highly expressed in other cells in the tumor microenvironment, specifically microglia and macrophages, how JAM-A expression in these cells affects tumor growth has yet to be determined. The goal of this study was to understand the role of microenvironmental JAM-A in mediating GBM growth. METHODS: Male and female wild-type (WT) and JAM-A-deficient mice were transplanted intracranially with the syngeneic glioma cell lines GL261 and SB28 and were assessed for differences in survival and microglial activation in tumors and in vitro. RNA-sequencing was performed to identify differentially regulated genes among all genotypes, and differences were validated in vitro and in vivo. RESULTS: We found that JAM-A-deficient female mice succumbed to GBM more quickly compared with WT females and JAM-A-deficient and male WT mice. Analysis of microglia in the tumors revealed that female JAM-A-deficient microglia were more activated, and RNA-sequencing identified elevated expression of Fizz1 and Ifi202b specifically in JAM-A-deficient female microglia. CONCLUSIONS: Our findings suggest that JAM-A functions to suppress pathogenic microglial activation in the female tumor microenvironment, highlighting an emerging role for sex differences in the GBM microenvironment and suggesting that sex differences extend beyond previously reported tumor cell-intrinsic differences.

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.

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.

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.

Adhering towards tumorigenicity: altered adhesion mechanisms in glioblastoma cancer stem cells.

Glioblastoma (GBM) is the most aggressive malignant primary brain tumor in adults with a high recurrence and mortality rate. GBM tumors contain a high degree of cellular heterogeneity, with cells exhibiting stem-like properties (cancer stem cells; CSCs) that are highly efficient at tumor initiation and are resistant to conventional therapies. CSCs interact with their tumor microenvironment by a large group of diverse cell adhesion molecules (CAMs) that participate in intercellular, intracellular and cell-extracellular matrix interactions. Despite the initial description of CAMs as tumor suppressors, recent work has highlighted specific CAMs that are essential for CSC maintenance and tumor progression. This review will highlight recent findings that provide support for a context-specific role of CAMs in CSC function and GBM progression.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling.

BACKGROUND: Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. METHODS: MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. RESULTS: Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. CONCLUSIONS: Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.