ALDH-positive Cancer Stem Cells Research Articles

Abstract A001: Silencing of EYA4 regulates stemness in H3K27M-mutant diffuse midline glioma

Abstract A001: Silencing of EYA4 regulates stemness in H3K27M-mutant diffuse midline glioma

Histone H3 K27M-mediated regulation of cancer cell stemness and differentiation in diffuse midline glioma

Therapeutic resistance remains a major obstacle to preventing progression of H3K27M-altered Diffuse Midline Glioma (DMG). Resistance is driven in part by ALDH-positive cancer stem cells (CSC), with high ALDH1A3 expression observed in H3K27M-mutant DMG biopsies. We hypothesized that ALDH-mediated stemness and resistance may in part be driven by the oncohistone itself. Upon deletion of H3K27M, ALDH1A3 expression decreased dramatically and was accompanied by a gain in astrocytic marker expression and a loss of neurosphere forming potential, indicative of differentiation. Here we show that the oncohistone regulates histone acetylation through ALDH1A3 in a Wnt-dependent manner and that loss of H3K27M expression results in sensitization of DMGs to radiotherapy. The observed elevated Wnt signaling in H3K27M-altered DMG likely stems from a dramatic suppression of mRNA and protein expression of the Wnt inhibitor EYA4 driven by the oncohistone. Thus, our findings identify EYA4 as a bona fide tumor suppressor in DMG that upon suppression, results in aberrant Wnt signaling to orchestrate stemness and differentiation. Future studies will explore whether overexpression of EYA4 in DMG can impede growth and invasion. In summary, we have gained mechanistic insight into H3K27M-mediated regulation of cancer stemness and differentiation, which provides rationale for exploring new therapeutic targets for DMG.

Candidates for early detection: Effect of pluripotent very small embryonic-like stem cells (VSELs) to transform into cancer stem cells (CSCs) and candidacy for early detection of cancer in a liquid biopsy.

e15024 Background: Prevailing somatic mutation theory (SMT) is mired with several paradoxes and still there is no clarity on how cancer initiates. No distinct pattern of somatic driver mutations specific to cancers affecting solid tissues has yet emerged even after massive NGS studies since 2007. Rather than cancer being a genetic disease, we postulate that endogenous, tissue-resident VSELs get transformed into CSCs to initiate cancer. Under normal conditions, VSELs along with tissue-specific progenitors work in a subtle manner to maintain life-long homeostasis in adult tissues. VSELs express steroid and gonadotropin hormone receptors, are directly vulnerable to environmental insults, and their dysfunction initiates various pathologies, including cancer. Recent mice studies have shown that environmental insults resulted in global hypomethylation, genomic instability and loss of imprinting (LOI) at IGF2/H19 loci that transformed quiescent VSELs into CSCs. Unlike VSELs, CSCs undergo excessive self-renewal, acquire bad-luck mutations during clonal expansion and oncogene activation to initiate cancers, and can also further mobilize to distant sites to cause metastasis. A 1000-sample clinical study based on OCT-4A expression by qRT-PCR suggested that CSCs that get mobilized from affected organs can serve as potential novel candidates for early detection of cancer through liquid biopsy. Methods: CSCs were enriched from the peripheral blood using our proprietary technology. They were enumerated by flow cytometry. VSELs undergo epigenetic changes and LOI to transform into CSCs. TaqMan based qRT-PCR was carried out to study OCT-4A and ALDH-1 positive CSCs and IGF2 expression to monitor LOI at the IGF2/H19 loci. LOI of IGF2 is commonly associated with initiation and also progression of multiple cancers and is also responsible for pushing VSELs out of quiescence and enter the cell cycle as CSCs. Results: Increased numbers of CSCs (epigenetically altered VSELs) were detected in peripheral blood samples of cancer subjects compared to normal subjects (p < 0.0001) by flow cytometry studies. The median absolute count of circulatory VSELs/CSCs was 190 per ml (IQR: 105-295) in normal subjects and 690 per ml (IQR: 370-1265) in cancer subjects. The qRT-PCR study conducted on 100 subjects showed increased expression of OCT-4A, IGF2 and ALDH1 genes in cancer compared to non-cancer subjects. Conclusions: Early detection of cancer by evaluating circulating tumor cells (CTCs) or tumor DNA (ctDNA) holds promise, but their scarce nature and mobilization in late stages only when tumor is > 3 mm in size, are major limitations for widespread translation. Circulating VSELs/CSCs in peripheral blood are evidently promising candidates to screen and classify subjects for cancer absence, likely risk, or presence.

Suppressing autophagy enhances disulfiram/copper-induced apoptosis in non-small cell lung cancer

Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.

Correlation between c-Met and ALDH1 contributes to the survival and tumor-sphere formation of ALDH1 positive breast cancer stem cells and predicts poor clinical outcome in breast cancer

c-Met is a receptor-type tyrosine kinase, which is involved in a wide range of cellular responses such as proliferation, motility, migration and invasion. It has been reported to be overexpressed in various cancers. However, the role of c-Met in breast cancer stem cells (CSCs) still remains unclear. We herein, show that c-Met expression is significantly elevated in Basal-like type of breast cancer in comparison with other subtypes. High expression of c-Met strongly correlated with the expression of two CSC markers, ALDH1A3 and CD133 in breast cancers. In addition, breast cancers at tumor stage III-IV expressing both c-Methigh and ALDH1A3high had poor prognosis. Furthermore, treatment with c-Met inhibitors (Crizotinib, Foretinib, PHA-665752 and Tivantinib) in MDA-MB157 cells with high c-Met protein expression resulted in significant suppression in cell viability, contrary to MDA-MB468 cells with low c-Met protein expression. These c-Met inhibitors also suppressed cell viability and tumor-sphere formation of ALDH1high breast cancer cells with high c-Met expression. These results suggest that c-Met in ALDH1 positive CSCs seems to play an important role in breast cancer repopulation. Therefore, we conclude that c-Met is a potential therapeutic target in ALDH1 positive breast CSCs.

Abstract 3895: Verrucarin J targets both cancer cells and cancer stem cells

Abstract Ovarian cancer is the fifth leading cause of death among women in the United States. The American Cancer Society estimates that approximately 22,280 women in the United States will be diagnosed with ovarian cancer and approximately 14,240 women will die as a result of ovarian cancer in 2016. The most common treatment for ovarian cancer is cytoreductive surgery, followed by chemotherapy (combination of carboplatin and paclitaxel). Although this regimen is initially effective in a high percentage of cases, unfortunately, within few months of initial treatment, tumor relapse occurs because of platinum-resistance. The presence of cancer stem cells (CSCs) are the main cause of this phenomenon. CSCs are a small population (2-5%) of cells in solid cancer which are chemo-resistant, highly tumorigenic, possess a self-renewal capacity, and the capability to differentiate into multiple lineage. Therefore, there is an urgent, unmet need for a more effective therapy that targets cancer cells as well as cancer stem cells. We screened for a novel drug that can target both cancer cells as well cancer stem cells and came cross Verrucarin J, a metabolite of the Myrothecium fungus family, which has not been tested for its antitumor activity. In our experiments, we treated two cisplatin-sensitive ovarian cancer cell lines (A2780 and OVCAR5) and one cisplatin-resistant cell line (A2780/CP70) with various concentrations of Verrucarin J. After treatment of cells, Verrucarin J showed: i) a highly significant inhibition of ovarian cancer cell proliferation in a dose- and time-dependent manner, with an IC50 value of approximately 10 nM after 48 h of treatment for all the three cell lines, ii) a significant increase in cell apoptosis, iii) a significant increase in DNA damage and generation of reactive oxygen species (ROS) compared to vehicle treated cells, iv) a significant down regulation of expression of cancer stem cells markers, and v) a significant inhibition of Notch1, Wnt1 and Shh signaling genes in a dose-dependent manner. Notch1, Wnt1 and Shh are major regulatory pathways for self-renewal and maintenance of cancer stem cells. Additionally, our experiments revealed that Verrucarin J inhibited the tumorigenic function (spheroid formation on ultra-low attachment plates) of isolated ALDH1 positive CSCs from ovarian cancer cell line A2780. Based on our results, we conclude that Verrucarin J is a highly potent anti-tumor drug and targets both cancer cells and cancer stem cells. Therefore, it may serve as a potential candidate for ovarian cancer treatment and replace currently used chemo-therapy. Note: This abstract was not presented at the meeting. Citation Format: Kelsey Carter, Seema Parte, Surinder K. Batra, Mariusz Z. Ratajczak, Sham S. Kakar. Verrucarin J targets both cancer cells and cancer stem cells [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 3895. doi:10.1158/1538-7445.AM2017-3895

Targeting ALDH1A1 by disulfiram/copper complex inhibits non-small cell lung cancer recurrence driven by ALDH-positive cancer stem cells.

The existence of cancer stem cells (CSCs) in non-small cell lung cancer (NSCLC) has profound implications for cancer therapy. In this study, a disulfiram/copper (DSF/Cu) complex was evaluated in vitro and in vivo for its efficacy to inhibit CSCs, which drive recurrence of NSCLC. First, we investigated whether DSF/Cu could inhibit ALDH-positive NSCLC stem cells in vitro and tumors derived from sorted ALDH-positive CSCs in vivo. DSF/Cu (0.5/1 μmol/l) significantly inhibited the expression of stem cell transcription factors (Sox2, Oct-4 and Nanog) and reduced the capacities of NSCLC stem cells for self-renewal, proliferation and invasion in vitro. Regular injections with DSF/Cu (60/2.4 mg/kg) reduced the size of tumors derived from sorted ALDH-positive stem cells. Two other NOD/SCID xenograft models were used to determine whether DSF/Cu could target NSCLC stem cells and inhibit tumor recurrence in vivo. DSF/Cu treatment eliminated ALDH-positive cells and inhibited tumor recurrence, which was reflected by reduced tumor growth in recipient mice that were inoculated with tumor cells derived from DSF/Cu-treated cells or primary xenografts. RNA interference and overexpression of ALDH isozymes suggested that ALDH1A1, which plays a key role in ALDH-positive NSCLC stem cells, might be the target of the DSF/Cu complex. Collectively, our data demonstrate that DSF/Cu targets ALDH1A1 to inhibit NSCLC recurrence driven by ALDH-positive CSCs. Thus, the DSF/Cu complex may represent a potential therapeutic strategy for NSCLC patients.

DOXIL when combined with Withaferin A (WFA) targets ALDH1 positive cancer stem cells in ovarian cancer

Ovarian cancer is a highly aggressive and deadly disease. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately, after few months of initial treatment, tumor relapse occurs due to platinum-resistance. DOXIL (liposomal preparation of doxorubicin) is a choice of drug for recurrent ovarian cancer. However, its response rate is very low and is accompanied by myocardial toxicity. Resistance to chemotherapy and recurrence of cancer is primarily attributed to the presence of cancer stem cells (CSCs), a small population of cells present in cancer. Effect of DOXIL and withaferin A (WFA), both alone and in combination, was investigated on cell proliferation of ovarian cancer cell line A2780 and tumor growth in SCID mice bearing i.p. ovarian tumors. ALDH1 cells were isolated from A2780 using cell sorter, and effect of DOXIL and WFA both alone and in combination on tumorigenic function of ALDH1 was studied using spheroids formation assays in vitro. Western blots were performed to examine the expression of ALDH1 and Notch 1 genes. In our studies, we showed, for the first time, that DOXIL when combined with withaferin A (WFA) elicits synergistic effect on inhibition of cell proliferation of ovarian cancer cells and inhibits the expression of ALDH1 protein, a marker for ALDH1 positive cancer stem cells (CSCs), and Notch1, a signaling pathway gene required for self-renewal of CSCs. Inhibition of expression of both ALDH1 and Notch1 genes by WFA was found to be dose dependent, whereas DOXIL (200 nM) was found to be ineffective. SCID mice, bearing i.p. ovarian tumors, were treated with a small dose of DOXIL (2 mg/kg) in combination with a sub-optimal dose of WFA (2 mg/kg) which resulted in a highly significant (60% to 70%) reduction in tumor growth, and complete inhibition of metastasis compared to control. In contrast, WFA treatment showed a significant reduction in tumor growth but no change in metastasis compared to control. DOXIL showed non-significant reduction in tumor growth and no change in metastasis compared to control. Isolated ALDH1 positive CSCs treated with the combination of DOXIL and WFA resulted in a significant reduction in spheroids formation (tumorigenic function of CSCs) and expression of ALDH1 protein. WFA when used alone at a concentration of 1.5 μM was found to be highly effective in suppression of ALDH1 expression, whereas DOXIL at a concentration of 200 nM was found to be ineffective. DOXIL in combination with WFA elicits synergistic effects, targets cancer stem cells, and has potential to minimize induction of drug resistance and reoccurrence of cancer. Based on our studies, we conclude that the combination of DOXIL with WFA has the potential to be an effective therapy for ovarian cancer and may ameliorate DOXIL related side effects as well as recurrence of ovarian cancer leading to increase in patients' survival rate.

Abstract 4797: VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% homology in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of mammary stem cells or tumor angiogenesis by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. VS-6063 is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing Phase 1/1b trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, VS-6063 also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by VS-6063 may more effectively suppress CSCs than inhibition of FAK alone. We find that human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. VS-6063 dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 release from human macrophages, while FAK siRNA did not. In the MDA-MB-231 breast cancer xenograft model in vivo, VS-6063 reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, VS-6063 substantially reduced the number of F4/80-positive macrophages in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by VS-6063 effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Format: Jonathan A. Pachter, Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Christian M. Vidal, Mahesh V. Padval, Qunli Xu. VS-6063 (defactinib) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2014-4797

Vs-6063 (Defactinib) Targets Mesothelioma Cancer Stem Cells Directly and Through Inhibition of Tumor-Associated Macrophages and Cytokine Production

ABSTRACT Aim: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase required for survival of cancer stem cells (CSCs). PYK2, the other FAK family member, shares 60% kinase domain homology. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma. Defactinib is a potent, orally active FAK kinase inhibitor that has completed phase I and is currently in clinical development in several indications including mesothelioma. Methods: Cytokine release from human macrophages was measured in response to small molecule FAK kinase inhibitors. Serum IL-6 and F4/80-positive macrophages were assessed by ELISA and immunofluorescence respectively. Results: In addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency. Using both FAK inhibitors and FAK siRNA, we demonstrated that FAK inhibition effectively reduces CSCs. Human monocyte-derived macrophages release IL-6 and IL-8, and these cytokines increased the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Accordingly, PYK2 siRNA decreased IL-6 and IL-8 release from human macrophages. In a xenograft model in vivo, defactinib reduced serum IL-6 while the FAK-only reference compound did not. Furthermore, defactinib substantially reduced the number of F4/80-positive macrophages in the tumors. Conclusions: These data suggest that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. Given the sensitivity of mesothelioma CSCs to defactinib, the use of this agent in a maintenance setting after first line chemotherapy may be an attractive strategy to extend survival of patients with mesothelioma. These data support the ongoing COMMAND study with defactinib in mesothelioma (Clinical trial NCT01870609). Disclosure: M. Keegan: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.E. Ring: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; V.N. Kolev: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; I.M. Shapiro: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; M.V. Padval: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; Q. Xu: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.; J.A. Pachter: Employee of Verastem, Inc. Stock Ownership in Verastem, Inc.

Abstract P2-09-14: Focal adhesion kinase (FAK) inhibitors VS-6063 and VS-4718 target breast cancer stem cells

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cellular signaling through integrins and growth factor receptors and functions in multiple steps of tumorigenesis. Both VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a Phase 1 clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. VS-6063 is currently being tested in multiple clinical studies both as a single agent and in combination with paclitaxel. VS-4718 was shown to inhibit tumor growth and metastasis in preclinical tumor models including triple negative breast cancer and is currently under evaluation in a Phase 1 clinical trial. We report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs) in vitro and in vivo in triple negative breast cancer models. VS-6063 and VS-4718 were evaluated in multiple cancer stem cell assays in vitro. Pre-treatment of SUM159 and MDA-MB-231 breast cancer cells with FAK inhibitors in matrigel reduced the percentage of ALDH-positive cells. The proportion of side population (SP) cancer stem cells was also reduced by FAK inhibition. The expression of ALDH1 in human breast tumor samples has previously been shown to correlate with poor prognosis (Ginestier et al 2007 Cell Stem Cell 1, 555). Significantly, ex vivo treatment of primary human breast tumor tissue with these FAK inhibitors also effectively reduced the proportion of ALDH-positive CSCs. Similar inhibitory effects on CSCs were observed in ovarian cancer cell lines OVCAR-5 and OVCAR-8. In direct contrast, cytotoxic agents such as paclitaxel and carboplatin increased the percentage of cancer stem cells in vitro, suggesting that these agents do not effectively target CSCs. Importantly, combination of either VS-6063 or VS-4718 with cytotoxic agents attenuated the enrichment of cancer stem cells induced by these cytotoxic agents. The in vivo effect of a FAK inhibitor on cancer stem cells was evaluated in an MDA-MB-231 human breast cancer orthotopic model. Oral administration of VS-4718 significantly reduced the presence of ALDH1-positive cells in tumors as assessed by immunofluorescence. Following dissociation of cells from tumors, CSCs were found to be reduced in VS-4718-treated tumors as measured by multiple assays. Importantly, cells isolated from VS-4718-treated tumors also showed reduced tumor-initiating capability upon re-implantation into immunodeficient mice in limiting dilutions. Our results provide clear demonstration that while FAK inhibitors significantly inhibit the growth of human breast tumor xenografts, they also preferentially target breast cancer stem cells in direct contrast to standard-of-care agents. These data provide a strong rationale for the clinical development of Verastem's FAK inhibitors in the treatment of breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-14.

Abstract P1-06-13: Periostin controls cytokine production to maintain breast cancer stem cells

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells (CSCs). Basal-like breast cancer represents a particularly aggressive disease subtype and the tumor cells share phenotypic similarities to breast cancer stem cells. However, the extracellular cues and intracellular pathways that CSCs rely on remain unclear. Working with a breast cancer progression model system we found the invasive breast cancer cell line to exhibit numerous stem-like characteristics. Gene expression profiling, along with a careful review of the literature, identified a secreted extracellular matrix molecule, periostin (POSTN), which we hypothesized to be required for the maintenance of a cancer stem cell state. POSTN expression was increased in numerous basal-like breast cancer cell lines, as well as in cells grown as mammospheres and in the CD44+/CD24- fraction of tumor cells. Furthermore, CSC-like lines displayed increased surface levels of the integrin alpha-v beta-3 heterodimer, a known receptor for POSTN, suggesting that hyperactivation of a POSTN signaling axis could potentially regulate the CSC state. Using stable cell lines expressing an shRNA directed at POSTN or ITGB3 we have found that POSTN signaling is required for efficient mammosphere formation and for the maintenance of an ALDH-positive CSC population. Disruption of this signaling axis resulted in decreased production of cytokines, including IL-6 and IL-8 - known regulators of CSCs - as well as downstream STAT3 signaling. Furthermore, high POSTN expression is associated with a worse clinical prognosis in ER-negative breast cancer. Therefore, we suggest that POSTN signaling through integrin receptors may enrich for highly tumorigenic breast CSCs in a subset of basal-like tumors, leading to aggressive and recurrent malignancies. This reveals a previously unknown link between POSTN signaling and cytokine production, and suggests that basal-like cancers can establish a microenvironmental niche supportive of cancer stem cells. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-13.

Abstract B283: Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production.

Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which mediates integrin and growth factor receptor signaling to regulate tumor growth, metastasis and angiogenesis. FAK is also required for the proliferation/survival of cancer stem cells (CSCs). Protein tyrosine kinase 2 (PYK2) is the most closely related protein kinase to FAK, sharing 60% amino acid identity in the kinase domain and a similar role in cellular signaling pathways. Several lines of evidence suggest that dual targeting of FAK and PYK2 should confer greater antitumor efficacy than an agent which only inhibits FAK. Inhibition of tumor angiogenesis or mammary stem cells by FAK inhibition can be bypassed by PYK2 activity, suggesting advantages of dual FAK/PYK2 inhibition. Accordingly, a critical role of PYK2 in CSC regulation and NSCLC patients’ prognosis has recently been reported (Kuang et al. 2013, Br J Cancer). Furthermore, preclinical data suggest that PYK2 inhibition can prevent growth of metastatic tumor cells in bone. Results of PYK2 knockout in mice suggest that PYK2 inhibition is likely to be well tolerated. Based on the observation of reduced macrophage infiltration in PYK2 knockout mice, we investigated effects of PYK2 inhibition on tumor-associated macrophages (TAMs). Abundance of TAMs has been correlated with poor prognosis in multiple cancer types including mesothelioma and breast cancer. Defactinib is a potent and orally active inhibitor of FAK kinase activity. Preliminary clinical activity has been observed in a Phase 1 single agent trial and in an ongoing trial in combination with paclitaxel in patients with ovarian cancer. We find that in addition to potent FAK inhibition, defactinib also inhibits PYK2 kinase activity with nanomolar potency in both biochemical enzymatic and cellular autophosphorylation assays. Using both pharmacological FAK inhibitors and FAK siRNA, we have previously demonstrated that FAK inhibition effectively reduces CSCs. We report here that dual targeting of FAK and PYK2 by defactinib may more effectively suppress CSCs than inhibition of FAK alone. We find that differentiated THP-1 macrophages release IL-6 and IL-8, and these cytokines increase the proportion of ALDH-positive CSCs in mesothelioma and breast cancer cell lines. Defactinib dose-dependently inhibited production of IL-6 and IL-8, whereas a FAK-only reference inhibitor had no effect on these cytokines. Furthermore, in MM87 mesothelioma and MDA-MB-231 breast cancer xenograft models, defactinib substantially reduced the number of F4/80-positive TAMs in the tumors. These data support a model in which TAMs release IL-6 and IL-8 in a PYK2-dependent manner. Dual inhibition of FAK and PYK2 by defactinib effectively decreases both the presence of TAMs in tumors and the ability of TAMs to release cytokines that stimulate CSC proliferation and survival, and may therefore provide the opportunity for more durable clinical response than inhibition of FAK alone. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B283. Citation Format: Jennifer E. Ring, Vihren N. Kolev, Irina M. Shapiro, Quentin G. Wright, Joseph R. Testa, Mahesh V. Padval, Qunli Xu, Jonathan A. Pachter. Defactinib (VS-6063) targets cancer stem cells directly and through inhibition of tumor-associated macrophages and cytokine production. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B283.

Clinicopathological Analysis of Breast Ductal Carcinoma in situ with ALDH1-Positive Cancer Stem Cells

Objective: The aim of this study was to elucidate the clinicopathological characteristics of breast ductal carcinomas in situ (DCIS) with aldehyde dehydrogenase 1 (ALDH1)-positive cancer stem cells (CSCs). Methods: DCIS (n = 194) were subjected to immunohistochemical staining and results were examined for associations with various clinicopathological parameters. The ALDEFLUOR assay for breast cancer cell lines was also performed to determine the proportion of CSCs according to intrinsic subtype. Results: DCIS with ALDH1-positive CSCs were significantly (p < 0.05) more likely to be estrogen receptor-α (ER)-negative, progesterone receptor (PgR)-negative, Ki67-positive and HER2-positive tumors. Luminal A subtype (8.6%) showed a significantly (p < 0.001) lower ALDH1 positivity than the other subtypes [luminal B (50.0%), luminal HER2 (36.8%), HER2 (35.3%) and triple-negative subtype (26.7%)]. Double immunostaining revealed that ALDH1-positive CSCs did not overlap with ER-, PgR- or Ki67-positive tumor cells but did overlap with HER2-positive tumor cells. The percentage of ALDEFLUOR-positive CSCs was lower in the luminal A cell lines (0.02% for T-47D and 0.4% for MCF7) than in the luminal HER2 (9.1% for BT-474 and 9.5% for MDA-MB-361), HER2 (13.9% for AU565 and 33.2% for SK-BR-3) and triple-negative cell lines (28.4% for MDA-MB-231 and 30.7% for MDA-MB-468). Conclusions: Our results suggest that most CSCs in DCIS are in the G0 phase (Ki67 negative) and do not express ER or PgR, but they do express HER2 in HER2-positive tumors.

Focal adhesion kinase (FAK) inhibitors VS-6063 and VS-4718 target cancer stem cells.

e13523 Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cellular signaling through integrins and growth factor receptors and functions in multiple steps of tumorigenesis. Both VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a phase I clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. VS-4718 is in late stage preclinical development and has been shown to inhibit tumor growth and metastasis in tumor models. We report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs). Methods: VS-6063 and VS-4718 were evaluated in the ALDEFLUOR and Side Population (SP) CSC assays in vitro and in xenograft tumors in vivo. Results: Treating SUM159 breast cancer cells with FAK inhibitors reduced the percentage of ALDEFLUOR+ and SP CSCs. Similar inhibitory effects on CSCs were observed in the ovarian cancer cell lines OVCAR-5 and OVCAR-8 and the mesothelioma cell line H2052. In direct contrast, cytotoxic agents paclitaxel, carboplatin and pemetrexed increased the percentage of CSCs, suggesting that these agents do not effectively target CSCs. Importantly, combination of either VS-6063 or VS-4718 with cytotoxic agents blocked induction of CSCs by these agents. The effect of a FAK inhibitor on CSCs in vivo was evaluated in an MDA-MB-231 human breast cancer orthotopic model. Oral administration of FAK inhibitor substantially reduced the number of ALDH1- positive cells in tumors as assessed by immunofluorescence. Following dissociation of cells from tumors, CSCs were found to be reduced in FAK inhibitor-treated tumors as measured by multiple assays. Furthermore, cells isolated from FAK inhibitor-treated tumors showed reduced tumor-initiating capability upon re-implantation into immunodeficient mice. FAK inhibitor also decreased ALDH1-positive CSCs in an orthotopic MM87 mesothelioma model. Conclusions: Our results provide clear demonstration that FAK inhibitors target cancer stem cells both in vitro and in xenograft tumors in vivo and support the planned clinical development of VS-6063 and VS-4718 to achieve durable clinical responses for cancer patients.

Abstract 4890: Periostin signaling regulates breast cancer stem cells.

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells (CSCs). However, the extracellular cues and intracellular pathways that CSCs rely on remain unclear. Working with a breast cancer progression model system we found the invasive breast cancer cell line to exhibit numerous stem-like characteristics. Gene expression profiling, along with a careful review of the literature, identified a secreted extracellular matrix molecule, periostin (POSTN), which we hypothesized to be required for the maintenance of a cancer stem cell state. POSTN expression was increased in numerous basal-like breast cancer cell lines that contain an expanded CSC population, as well as in cells grown as mammospheres and in the CD44+/CD24- fraction of tumor cells. Furthermore, CSC-like lines displayed increased surface levels of the alpha-v beta-3 integrin heterodimer, a known receptor for POSTN, suggesting that hyperactivation of a POSTN signaling axis could potentially regulate the CSC state. Using stable cell lines expressing an shRNA directed at POSTN we have found that POSTN is required for efficient mammosphere formation and for the maintenance of an ALDH-positive CSC population. In line with this, we found these cells had impaired Erk and Wnt signaling and expressed less IL-6 and IL-8, known regulators of CSCs. Furthermore, high POSTN expression correlates with a worse clinical prognosis in basal-like breast cancer patients. Therefore, we suggest that POSTN signaling through integrin receptors may enrich for highly tumorigenic breast CSCs in a subset of basal-like tumors, leading to aggressive and recurrent malignancies. More broadly, our results imply that a supportive tumor niche can be established and maintained by cancer cells, and raises the possibility of targeting components of this extracellular environment to eradicate breast CSCs. Citation Format: Arthur W. Lambert, Sait Ozturk, Panos Papageorgis, Hamid Abdolmaleky, Sam Thiagalingam. Periostin signaling regulates breast cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4890. doi:10.1158/1538-7445.AM2013-4890