Diana J. Azzam

Exosome Transfer from Stromal to Breast Cancer Cells Regulates Therapy Resistance Pathways

Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth.

VEGF drives cancer-initiating stem cells through VEGFR-2/Stat3 signaling to upregulate Myc and Sox2

Vascular endothelial growth factor-A (VEGF), a potent angiogenic factor, is also implicated in self-renewal in several normal tissue types. VEGF has been shown to drive malignant stem cells but mechanisms thereof and tumor types affected are not fully characterized. Here, we show VEGF promotes breast and lung cancer stem cell (CSC) self-renewal via VEGF receptor-2 (VEGFR-2)/STAT3-mediated upregulation of Myc and Sox2. VEGF increased tumor spheres and aldehyde dehydrogenase activity, both proxies for stem cell function in vitro, in triple-negative breast cancer (TNBC) lines and dissociated primary cancers, and in lung cancer lines. VEGF exposure before injection increased breast cancer-initiating cell abundance in vivo yielding increased orthotopic tumors, and increased metastasis from orthotopic primaries and following tail vein injection without further VEGF treatment. VEGF rapidly stimulated VEGFR-2/JAK2/STAT3 binding and activated STAT3 to bind MYC and SOX2 promoters and induce their expression. VEGFR-2 knockdown or inhibition abrogated VEGF-mediated STAT3 activation, MYC and SOX2 induction and sphere formation. Notably, knockdown of either STAT3, MYC or SOX2 impaired VEGF-upregulation of pSTAT3, MYC and SOX2 expression and sphere formation. Each transcription factor, once upregulated, appears to promote sustained activation of the others, creating a feed-forward loop to drive self-renewal. Thus, in addition to angiogenic effects, VEGF promotes tumor-initiating cell self-renewal through VEGFR-2/STAT3 signaling. Analysis of primary breast and lung cancers (>1300 each) showed high VEGF expression, was prognostic of poor outcome and strongly associated with STAT3 and MYC expression, supporting the link between VEGF and CSC self-renewal. High-VEGF tumors may be most likely to escape anti-angiogenics by upregulating VEGF, driving CSC self-renewal to re-populate post-treatment. Our work highlights the need to better define VEGF-driven cancer subsets and supports further investigation of combined therapeutic blockade of VEGF or VEGFR-2 and JAK2/STAT3.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b–Mediated Malignant Progression

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ-secretase inhibitor drug responses

Increasing evidence suggests that stem‐like cells mediate cancer therapy resistance and metastasis. Breast tumour‐initiating stem cells (T‐ISC) are known to be enriched in CD44+CD24neg/low cells. Here, we identify two T‐ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44+CD24low+ subpopulation generates CD44+CD24neg progeny with reduced sphere formation and tumourigenicity. CD44+CD24low+ populations contain subsets of ALDH1+ and ESA+ cells, yield more frequent spheres and/or T‐ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA‐MB‐231 model, metastatic potential. CD44+CD24low+ but not CD44+CD24neg express activated Notch1 intracellular domain (N1‐ICD) and Notch target genes. We show N1‐ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44+CD24low+cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44+CD24low+ cells, but CD44+CD24neg were resistant. While GSI hold promise for targeting T‐ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T‐ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti‐cancer drug responsiveness.

Src Inhibition with Saracatinib Reverses Fulvestrant Resistance in ER-Positive Ovarian Cancer Models In Vitro and In Vivo

Purpose: More effective, less toxic treatments for recurrent ovarian cancer are needed. Although more than 60% of ovarian cancers express the estrogen receptor (ER), ER-targeted drugs have been disappointing due to drug resistance. In other estrogen-sensitive cancers, estrogen activates Src to phosphorylate p27 promoting its degradation and increasing cell-cycle progression. Because Src is activated in most ovarian cancers, we investigated whether combined Src and ER blockade by saracatinib and fulvestrant would circumvent antiestrogen resistance. Experimental Design: ER and Src were assayed in 338 primary ovarian cancers. Dual ER and Src blockade effects on cell cycle, ER target gene expression, and survival were assayed in ERα+ ovarian cancer lines, a primary human ovarian cancer culture in vitro, and on xenograft growth. Results: Most primary ovarian cancers express ER. Src activity was greater in ovarian cancer lines than normal epithelial lines. Estrogen activated Src, ER-Src binding, and ER translocation from cytoplasm to nucleus. Estrogen-mediated mitogenesis was via ERα, not ERβ. While each alone had little effect, combined saracatinib and fulvestrant increased p27 and inhibited cyclin E-Cdk2 and cell-cycle progression. Saracatinib also impaired induction of ER-target genes c-Myc and FOSL1; this was greatest with dual therapy. Combined therapy induced autophagy and more effectively inhibited ovarian cancer xenograft growth than monotherapy. Conclusions: Saracatinib augments effects of fulvestrant by opposing estrogen-mediated Src activation and target gene expression, increasing cell-cycle arrest, and impairing survival, all of which would oppose antiestrogen resistance in these ER+ ovarian cancer models. These data support further preclinical and clinical evaluation of combined fulvestrant and saracatinib in ovarian cancer. Clin Cancer Res; 18(21); 5911–23. ©2012 AACR.

Combined Src and ER blockade impairs human breast cancer proliferation in vitro and in vivo

Antiestrogen therapies arrest susceptible estrogen receptor (ER)-positive breast cancers by increasing p27. Since Src phosphorylates p27 to promote p27 proteolysis, Src activation observed in up to 40% of ER-positive cancers may contribute to antiestrogen resistance. In this article, we show that treatment with the Src-inhibitor saracatinib (AZD0530) together with ER-blocking drugs increased breast cancer cell cycle arrest via p27. Saracatinib and fulvestrant together more effectively increased p27, reduced Ki67, and impaired MDA-MB-361 xenograft tumor growth in vivo than either of the drugs alone. In contrast, saracatinib monotherapy rapidly gave rise to drug resistance. Since combined ER and Src inhibition delays development of resistance in vivo, these data support further clinical investigation of saracatinib in combination with fulvestrant for women with ER-positive breast cancer. Proteomic analysis revealed striking bypass activation of the mTOR pathway in saracatinib-resistant tumors. mTORC1 activation also arose following long-term culture of ER-positive breast cancer lines in the presence of saracatinib. These data indicate the utility of proteomic analysis of drug-resistant tumors to identify potential means of drug resistance. The use of mTOR kinase inhibitors with saracatinib may subvert drug resistance and prove to be more effective than saracatinib alone.

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells.” In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.

Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells

Purpose: Rational targeted therapies are needed for treatment of ovarian cancers. Signaling kinases Src and MAPK are activated in high-grade serous ovarian cancer (HGSOC). Here, we tested the frequency of activation of both kinases in HGSOC and the therapeutic potential of dual kinase inhibition. Experimental Design: MEK and Src activation was assayed in primary HGSOC from The Cancer Genome Atlas (TGGA). Effects of dual kinase inhibition were assayed on cell-cycle, apoptosis, gene, and proteomic analysis; cancer stem cells; and xenografts. Results: Both Src and MAPK are coactivated in 31% of HGSOC, and this associates with worse overall survival on multivariate analysis. Frequent dual kinase activation in HGSOC led us to assay the efficacy of combined Src and MEK inhibition. Treatment of established lines and primary ovarian cancer cultures with Src and MEK inhibitors saracatinib and selumetinib, respectively, showed target kinase inhibition and synergistic induction of apoptosis and cell-cycle arrest in vitro, and tumor inhibition in xenografts. Gene expression and proteomic analysis confirmed cell-cycle inhibition and autophagy. Dual therapy also potently inhibited tumor-initiating cells. Src and MAPK were both activated in tumor-initiating populations. Combination treatment followed by drug washout decreased sphere formation and ALDH1+ cells. In vivo, tumors dissociated after dual therapy showed a marked decrease in ALDH1 staining, sphere formation, and loss of tumor-initiating cells upon serial xenografting. Conclusions: Selumetinib added to saracatinib overcomes EGFR/HER2/ERBB2–mediated bypass activation of MEK/MAPK observed with saracatinib alone and targets tumor-initiating ovarian cancer populations, supporting further evaluation of combined Src–MEK inhibition in clinical trials. Clin Cancer Res; 24(19); 4874–86. ©2018 AACR.

Abstract 1906: The role of immunogenic SPANX antigens in distinct cancer stem cell subsets within triple negative breast cancers

Breast cancer is the leading cause of death in women, primarily due to metastatic disease rather than the primary tumor. Median survival with metastatic breast cancer is 3 years, with no statistically significant change in survival in over 20 years. Triple-negative breast cancer (TNBC) lacks estrogen and progesterone receptor expression and ERBB2 amplification and is the most lethal form of breast cancer. It is resistant to endocrine therapy and chemo-resistance and metastasis invariably emerge. Increasing evidence indicates that cancer stem cells (CSCs) are chemo-resistant and initiate metastasis. We have previously identified and characterized distinct subsets of CSCs within TNBC cell lines and patient-derived tumors. Within the surface CD44+ populations, CD24 expression defines two subsets of CSCs: mesenchymal-like CD24neg and epithelial-like CD24low+. Epithelial-like CD24low+ cells are more aggressive than CD24neg cells, with increased self-renewal, migration, invasion, tumor-initiation and metastatic potential. In addition, CD24low+ cells are enriched following chemo- and radiation therapies; greater than 80% of CD24neg cells die and the majority of cells that survive are CD24low+. Hence, therapies that selectively eliminate the CD24low+ population in TNBC have the potential to be of enormous benefit to cancer patients. Therapeutic targeting of surface CD24 using antibodies have not been successful due to the expression of CD24 on many different cell types including B cells. Thus, there is a need for more selective strategies to target CSC populations without affecting normal cells. Here, we show for the first time, increased expression of a family of cancer/testis antigens (CTAs) namely SPANX in the most aggressive CD24low+ CSC population. CTAs belong to a class of testis-derived proteins which are only expressed in germ cells in the male testis, and the expression of CTA genes is entirely silenced in the adult somatic tissues. Thus, SPANX may serve as a selective marker for targeting CSCs. In addition, we demonstrate a functional role for SPANX in mediating CSC phenotype. Knockdown of SPANX decreased the percent of CD24low+ CSCs in TNBCs. It also attenuated the proliferation, migration and invasion of CD24low+ cells. Radiation treatment increased SPANX expression levels and enriched for CD24low+ cells. Loss of SPANX resulted in increased cell death of CD24low+ cells after radiation treatment. Hence, we show that SPANX may be promoters of the most aggressive CSC subset of TNBC. Since SPANX is highly immunogenic, our data provide rationale for further testing of combined radiation and immunotherapy approaches in the treatment of this deadly cancer. It also supports the use of protective and therapeutic SPANX vaccines against the most aggressive CSC subset in TNBC. Citation Format: Lauren Shahin, Elena Cubedo, Ebony Coats, Jeff Boyd, Diana Azzam. The role of immunogenic SPANX antigens in distinct cancer stem cell subsets within triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1906. doi:10.1158/1538-7445.AM2017-1906

Abstract POSTER-THER-1431: Combined Src and MEK inhibition decreases ovarian cancer (OVCA) cell growth, tumorigenicity and aldehyde dehydrogenase positive tumor-initiating-cells

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Introduction: More effective treatments for recurrent OVCA are needed. Src kinase and the MAPK pathway are activated in OVCAs. Saracatinib, is a potent inhibitor of Abl and Src family kinases and selumetinib, is a potent, selective, ATP-noncompetitive inhibitor of MEK 1/2 kinases. Our earlier work has shown that resistance to saracatinib monotherapy emerges in vitro and in vivo. Since oncogenic activation of both Src and MEK pathways has been observed in OVCAs and because resistance to Src inhibition emerges with monotherapy, we hypothesized that blocking both kinases with combination therapy will more effectively inhibit OVCA cell proliferation than either drug alone. Experimental Design: Effects of dual Src and MEK blockade on cell cycle, autophagy, apoptosis and xenografts were assayed. Drug effects were confirmed in primary human OVCA tumor cells. Drug effects were evaluated by RPPA and gene expression analysis by microarray. Cells enriched for stem cell marker, aldehyde dehydrogenase 1 (ALDH1+) were sorted by FACS. Drug effects on ALDH1+ cells, and sphere formation were analyzed. Xenografts after treatment were dissociated and transplanted in vivo in a limiting dilution. Results: Analysis of TCGA high grade serous OVCAs by RPPA showed the majority express pSrc (T419) (n=300/408) and pMAPK (n=310/408) and patients with high pSrc /pMAPK expression had a poorer survival than those with low pSrc /pMAPK (p=0.004). Increased phosphorylation of MAPK was observed with prolonged saracatinib treatment. While each drug alone caused partial growth arrest, treatment with both saracatinib and selumetinib, decreased Src, MAPK, and Akt and caused synergistic cell cycle arrest, which was mediated by p27 binding to cyclin E/cdk2. Gene expression enrichment analysis showed the top 20 gene sets dowregulated by combination treatment involved cell cycle and the gene set most upregulated was autophagy. RPPA confirmed GEA and demonstrated that combination treatment decreased not only Src, MAPK pathways but the PI3K/AKT pathway. Combination treatment induced apoptosis and autophagy. Src and MEK phosphorylation was increased in ALDH1+ sorted populations and combination treatment decreased %ALDH1+cells and % spheres. Combination treatment was more effective in vivo than monotherapy. Transplantation of dissociated cells after treatment in a limiting dilution in vivo resulted in decreased tumor formation and stem cell frequency in the combination treated grp (p=0.01). Conclusion: MEK inhibition augments the effects of saracatinib by blocking bypass activation of the MAPK and PI3K/AKT pathway, increasing cell cycle arrest, impairing survival and targeting self-renewing subpopulations. These data support further pre-clinical and clinical evaluation of combined saracatinib and MEK inhibition in OVCA. Citation Format: F. Simpkins, K. Hew, D. Azzam, Z. Wei, G. Zhang, J. Sun, D. Zhang, K. Jang, W. Liu, Y. Lu, G. Mills, I. Tan, J. Slingerland. Combined Src and MEK inhibition decreases ovarian cancer (OVCA) cell growth, tumorigenicity and aldehyde dehydrogenase positive tumor-initiating-cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1431.