Cancer Stem Cell Pool Research Articles

Abstract 2891: Retinoic acid directs breast cancer cell state changes through regulation of TET2-aPKC pathway

Abstract The key molecular mechanism governing the cancer cell state (stem cell-like state vs. differentiation state) to control the cancer stem cell (CSC) pool remains elusive. This study provides the first evidence showing that all-trans retinoic acid (ATRA) induces the interaction and chromatin recruitment of a novel RARB-TET2 complex to epigenetically activate a specific cohort of gene targets, including MiR-200c. TET2-activated miR-200c further targets and suppresses aPKC, a cell polarity protein that plays a pivotal role in directing asymmetric division of mammalian stem cells to sustain the stem cell pool. Our data reveals that pharmacological concentration of ATRA effectively down-regulates aPKC through activation of miR-200c, leading to a decrease of the stem cell-like populations from non-tumorigenic mammary epithelial cells and non-aggressive breast cancer cells. However, aggressive breast cancer cells that manifest TET2-miR200c dysregulation sustain a CSC pool highly resistant to ATRA, where inhibition of aPKC directs the resistant CSCs to the luminal cell-like state and sensitization to Tamoxifen, resulting in abrogation of mammary tumor growth and progression. Together, these findings elucidate a novel RARB-TET2-miR200c-aPKC signaling pathway that directs cancer cell state changes and also provide previously unidentified therapeutic implications for aPKC inhibitors in diminishment of breast CSCs to eradicate breast cancer. Citation Format: Meng-Ju Wu, Chun-Ju Chang. Retinoic acid directs breast cancer cell state changes through regulation of TET2-aPKC pathway [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 2891. doi:10.1158/1538-7445.AM2017-2891

Abstract 1268: Bitter melon efficacy against human pancreatic cancer cells: possible involvement of cellular stemness and metabolome targets

Abstract Pancreatic cancer (PanC) has a dismal 5-year survival rate of < 5%. Symptomless progression, late diagnosis and rising drug resistance to leading chemotherapeutic agents like gemcitabine, add to PanC severity. Hence, there is a critical requirement for identifying alternative novel non-toxic agents (dietary/non-dietary) aimed at effective management of PanC with minimal patient distress. Bitter melon (Momordica charantia), a dietary agent, is actively being investigated for its anti-cancer potential against a variety of malignancies, including our work in PanC. Recent studies by us have shown bitter melon juice (BMJ) efficacy against a panel of human PanC cell lines in culture and tumor xenografts. For mechanistic studies, here we evaluated the efficacy of BMJ against cancer stem cells (CSCs) pool in PanC cells. Results indicated that BMJ treatment (0.25-2% v/v) significantly decreased CSC enriched (CD44+CD24+EpCAMhigh) PanC cell population. Successive immunofluorescence/ immunohistochemical analysis of BMJ exposed cells, spheroids and MiaPaCa2 xenograft tumors showed a significant reduction in CSC markers/ regulatory molecules. Since cancer cell metabolism is an important component to be targeted to control cancer growth and progression, we next assessed BMJ efficacy in that direction. Indeed, BMJ mediated its anti-PanC effect via activating the key metabolic regulator AMPK. Concomitantly, to determine whether BMJ-effects were nutrient/energy stress dependent, we next sought to determine the cellular energy profile and major metabolic changes induced by BMJ. PanC cell metabolome analysis was done, as a function of time, to determine the shift in PanC cell metabolism on BMJ exposure. 1H-, 13C- and 31P-NMR spectra of PanC cell monolayers treated with BMJ (2% v/v) at early ( 4h) and late (72h) time points showed significantly altered energy status and modulation of major metabolic pathways involved in PanC cell growth and proliferation. A significant reduction in glucose uptake and cellular energy levels (ATP/ADP) ratio was observed, but lactate export was increased. BMJ also modulated nucleotides and aromatic amino acids involved in protein precursors/synthesis for PanC growth and proliferation. Total fatty acids and phosphatidylcholine levels (a precursor of membrane lipids) were also downregulated by BMJ. Glutamine and glutamate levels were constrained by BMJ, indicating that it could potentially limit PanC cell ability for targeting an alternative energy source. Furthermore, the cellular levels and membrane localization of GLUT1 (major glucose transporter overexpressed in PanC) were also suppressed after BMJ treatment. Collectively, these results suggest that BMJ targets PanC CSCs and cellular metabolism in its efficacy against this deadly malignancy (supported by CA195708). Citation Format: Deepanshi Dhar, Gagan Deep, Sushil Kumar, Chapla Agarwal, Natalie Serkova, Michael Wempe, Komal Raina, Rajesh Agarwal. Bitter melon efficacy against human pancreatic cancer cells: possible involvement of cellular stemness and metabolome targets [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 1268. doi:10.1158/1538-7445.AM2017-1268

Abstract 1915: Deconstructing cancer stem cells: IL-6, G-CSF and Activin-A as mediators of stroma-orchestrated epithelial cells’ dedifferentiation

Abstract Intercellular communication in cancer is a deceiving and extremely efficient process through which the corporal machinery is hijacked by a panoply of cellular and molecular strategies turning the entire human body into an evolutionary arena. The extensive crosstalk mediated by cytokines and chemokines overcome the inefficiency of the invasion-metastasis cascade, thus allowing the development of the often-fatal metastatic disease. Cancer stem cells (CSCs) have recently been implicated in major steps of the tumorigenic process, namely in tumor initiation, metastasis formation and tumor relapse following therapy. Yet, despite their relevance, the exact mechanisms underlying their formation are still unclear. After observing that the malignant human bronchial epithelial RenG2 cells dedifferentiated following culture in the subcutaneous mouse lumbar region, co-cultures of surgically isolated mice lumbar stromal cells with RenG2 cells were established and the conditioned media studied by multiplex and ELISA. Consequently, Interleukin-6 (IL-6), Granulocyte colony-stimulating factor (G-CSF) and Activin-A were identified as the paracrine mediators of the intercellular communication process. Aiming to ascertain the individual role of each cytokine in the dedifferentiation process, as well as to access the involvement of exosomes as transport vehicle, the same co-cultures were reproduced in the presence of specific cytokine-communication blockers, either individually or in combinations of up to three blockers, and exosome-mediated communication inhibitors. Finally, exosomes were also collected from control co-cultures and their cargo screened for the target cytokines. ELISA showed that the three cytokines were present inside fibroblasts-secreted exosomes. Moreover, whenever exosomes’ release was blocked, dedifferentiation was abrogated, further proving the role of the aforementioned cytokines and of exosomes in the dedifferentiation process. Additionally, the cytokine-blocking experiments revealed that only IL-6 and Activin-A were endowed with the potential to orchestrate dedifferentiation, as when at least one of these cytokines was present a stem cell population developed inside RenG2 cells. Finally, G-CSF appeared to be decisive in maintaining the undifferentiated phenotype, as a larger pool of CSCs was attained whenever this cytokine and either IL-6 or Activin-A were present. Altogether the attained results implicated IL-6 and Activin-A in the formation of CSCs by dedifferentiation, and G-CSF as a potent keeper of the dedifferentiation status. Subsequent studies are now required to access the use of these cytokines as therapeutic targets so the tumorigenic process may be abrogated in its initial steps, improving patients’ prognosis and survival. Work sponsored by FEDER, POFC-COMPETE and the FCT grants PTDC/BBB-BQB/2450/2012 and SFRH/BD/33884/2009. Citation Format: Carlos F. Rodrigues, Eurico Serrano, Marco Cunha, Maria I. Patrício, Mariana Val, João Fonseca, Célia Gomes, Antero Abrunhosa, Artur Paiva, Filomena Botelho, Lina Carvalho, Isabel M. Carreira, Alpoim Carmen. Deconstructing cancer stem cells: IL-6, G-CSF and Activin-A as mediators of stroma-orchestrated epithelial cells’ dedifferentiation [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 1915. doi:10.1158/1538-7445.AM2017-1915

Abstract 1461: miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative breast cancer

Abstract Introduction: Meta-analysis shows women with diabetes have a 20% increased risk of breast cancer. Diabetes increases the risk of breast cancer mortality. Additionally, patients with breast cancer and preexisting diabetes have an increased risk for distant metastasis compared with non-diabetic counterparts. The molecular mechanisms for distant metastasis in breast cancer patients with diabetes are not very well understood. Our preliminary data suggested that hyperglycemia enhances stem cell activity in triple negative breast cancer (TNBC) cells. Here, we report miR-424 to be a key regulator of breast cancer stem cell pool dynamics under hyperglycemic environment. Materials and Methods: MDA-MB-231, TNBC cell line was maintained under euglycemic (5mM) and hyperglycemic (10mM) culture conditions. RT-PCR based microRNA array was performed followed by validation of significantly altered microRNA to confirm the effect of hyperglycemia on the global microRNA profile. Flow cytometric analysis for CD44+/CD24- was performed to assess the alterations in breast cancer stem cell population. In addition, sphere-forming assay were also performed. Mir-424 over-expressing or knocked down cell lines were established from parental MDA-MB-231 cells and maintained in hyperglycemic or euglycemic condition respectively. Promoter analysis for PR/SET domain 14 (prdm14) was done. Western blotting and immunofluorescence assays were performed for cdc42 (cell division control 42), pStat5 (phospho signal transducer and activator of transcription factor 5) and prdm14. Results: Loss of miR-424 in TNBC cells under euglycemic conditions led to enhanced stem cell activity; whereas over expression of miR- 424 under hyperglycemic conditions resulted in suppressed stem cell activity. Introduction of cdc42, (a miR-424 target gene) in miR-424 over expressing TNBC cells under hyperglycemic conditions leads to increased stem cell activity demonstrating the significance of miR-424-cdc42 signaling in hyperglycemia. Over-expression of miR-424 in TNBC dramatically reduced its metastatic abilities in vivo. Mechanistically, we found that miR-424-cdc42 signaling in hyperglycemia promotes Prdm14 activation, a stem cell regulator through increased phosphorylation of Stat5. Conclusions: Our findings establish a key molecular signaling cascade (miR-424→cdc42→prdm14) that promotes TNBC stem cell activity under hyperglycemic conditions. Citation Format: Sushmita B. Nandy, Alexis Orozco, Gautham Prabhakar, Viktoria Stewart, Stephanie Jones, Paloma Munoz, Ramadevi Subramani, Diego Pedroza, Rajkumar Lakshmanaswamy. miR-424-cdc42, key signaling axis in hyperglycemic regulation of stemness in triple negative breast cancer [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 1461. doi:10.1158/1538-7445.AM2017-1461

Natural and synthetic progestins enrich cancer stem cell-like cells in hormone-responsive human breast cancer cell populations in vitro.

Clinical trials and studies have shown that combination estrogen/progestin hormone replacement therapy, but not estrogen therapy alone or placebo, increases breast cancer risk in postmenopausal women. Using animal models, we have previously shown that both natural and synthetic progestins (including medroxyprogesterone acetate [MPA], a synthetic progestin used widely in the clinical setting) accelerate the development of breast tumors in vivo and increase their metastasis to lymph nodes. Based on these observations, we have hypothesized that progestin-induced breast cancer tumor growth and metastasis may be mediated by an enrichment of the cancer stem cell (CSC) pool. In this study, we used T47-D and BT-474 hormone-responsive human breast cancer cells to examine the effects of progestin on phenotypic and functional markers of CSCs in vitro. Both natural and synthetic progestins (10 nM) significantly increased protein expression of CD44, an important CSC marker in tumor cells. MPA increased the levels of both CD44 variants v3 and v6 associated with stem cell functions. This induction of CD44 was blocked by the antiprogestin RU-486, suggesting that this process is progesterone receptor (PR) dependent. CD44 induction was chiefly progestin dependent. Because RU-486 can bind other steroid receptors, we treated PR-negative T47-DCO-Y cells with MPA and found that MPA failed to induce CD44 protein expression, confirming that PR is essential for progestin-mediated CD44 induction in T47-D cells. Further, MPA treatment of T47-D cells significantly increased the activity of aldehyde dehydrogenase (ALDH), another CSC marker. Finally, two synthetic progestins, MPA and norethindrone, significantly increased the ability of T47-D cells to form mammospheres, suggesting that enrichment of the CD44high, ALDHbright subpopulation of cancer cells induced by MPA exposure is of functional significance. Based on our observations, we contend that exposure of breast cancer cells to synthetic progestins leads to an enrichment of the CSC pool, supporting the development of progestin-accelerated tumors in vivo.

Targeting KDM1A attenuates Wnt/β-catenin signaling pathway to eliminate sorafenib-resistant stem-like cells in hepatocellular carcinoma

Use of the tyrosine kinase inhibitor sorafenib in patients with advanced hepatocellular carcinoma (HCC) is often hindered by the development of resistance, which has been recently shown to be associated with the emergence of a cancer stem cell (CSC) subpopulation. However, it remains largely unknown whether epigenetic mechanisms, especially histone posttranslational modifications, are causally linked to the maintenance of stem-like properties in sorafenib-resistant HCC. In this study, we report that the activity of lysine-specific histone demethylase 1A (KDM1A or LSD1) is required for the emergence of cancer stem cells following prolonged sorafenib treatment. As such, KDM1A inhibitors, such as pargyline and GSK2879552, dramatically suppress stem-like properties of sorafenib-resistant HCC cells. Mechanistically, KDM1A inhibitors derepress the expression of multiple upstream negative regulators of the Wnt signaling pathway to downregulate the β-catenin pathway. More importantly, KDM1A inhibition resensitizes sorafenib-resistant HCC cells to sorafenib in vivo, at least in part through reducing a CSC pool, suggesting a promising opportunity for this therapeutic combination. Together, these findings suggest that KDM1A inhibitors may be utilized to alleviate acquired resistance to sorafenib, thus increasing the therapeutic efficacy of sorafenib in HCC patients.

Pre‐clinical validation of a selective anti‐cancer stem cell therapy for Numb‐deficient human breast cancers

The cell fate determinant Numb is frequently downregulated in human breast cancers (BCs), resulting in p53 inactivation and an aggressive disease course. In the mouse mammary gland, Numb/p53 downregulation leads to aberrant tissue morphogenesis, expansion of the stem cell compartment, and emergence of cancer stem cells (CSCs). Strikingly, CSC phenotypes in a Numb‐knockout mouse model can be reverted by Numb/p53 restoration. Thus, targeting Numb/p53 dysfunction in Numb‐deficient human BCs could represent a novel anti‐CSC therapy. Here, using patient‐derived xenografts, we show that expansion of the CSC pool, due to altered self‐renewing divisions, is also a feature of Numb‐deficient human BCs. In these cancers, using the inhibitor Nutlin‐3 to restore p53, we corrected the defective self‐renewal properties of Numb‐deficient CSCs and inhibited CSC expansion, with a marked effect on tumorigenicity and metastasis. Remarkably, a regimen combining Nutlin‐3 and chemotherapy induced persistent tumor growth inhibition, or even regression, and prevented CSC‐driven tumor relapse after removal of chemotherapy. Our data provide a pre‐clinical proof‐of‐concept that targeting Numb/p53 results in a specific anti‐CSC therapy in human BCs.

A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer.

Cancer stem cells (CSCs) have been hypothesized to represent the driving force behind tumour progression and metastasis, making them attractive cancer targets. However, conclusive experimental evidence for their functional relevance is still lacking for most malignancies. Here we show that the leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) identifies intestinal CSCs in mouse tumours engineered to recapitulate the clinical progression of human colorectal cancer. We demonstrate that selective Lgr5+ cell ablation restricts primary tumour growth, but does not result in tumour regression. Instead, tumours are maintained by proliferative Lgr5- cells that continuously attempt to replenish the Lgr5+ CSC pool, leading to rapid re-initiation of tumour growth upon treatment cessation. Notably, CSCs are critical for the formation and maintenance of liver metastasis derived from colorectal cancers. Together, our data highlight distinct CSC dependencies for primary versus metastasic tumour growth, and suggest that targeting CSCs may represent a therapeutic opportunity for managing metastatic disease.

Retinoic acid directs breast cancer cell state changes through regulation of TET2-PKCζ pathway

The key molecular mechanism governing the cancer cell state (stem cell-like state vs differentiation state) to control the cancer stem cell (CSC) pool remains elusive. This study provides the first evidence showing that all-trans retinoic acid (ATRA) induces the interaction and chromatin recruitment of a novel RARβ-TET2 complex to epigenetically activate a specific cohort of gene targets, including MiR-200c. TET2-activated miR-200c further targets and suppresses PKCζ, a cell polarity protein that has a pivotal role in directing asymmetric division of mammalian stem cells to sustain the stem cell pool. Our data reveal that pharmacological concentration of ATRA effectively downregulates PKCζ through activation of miR-200c, leading to a decrease of the stem cell-like populations from non-tumorigenic mammary epithelial cells and non-aggressive breast cancer cells. However, aggressive breast cancer cells that manifest TET2-miR-200c dysregulation sustain a CSC pool highly resistant to ATRA, where inhibition of PKCζ directs the resistant CSCs to the luminal cell-like state and sensitization to tamoxifen, resulting in abrogation of mammary tumor growth and progression. Together, these findings elucidate a novel RARβ-TET2-miR-200c-PKCζ signaling pathway that directs cancer cell state changes and also provide previously unidentified therapeutic implications for PKCζ inhibitors in diminishment of breast CSCs to eradicate breast cancer.

Abstract P3-06-09: Selection of presumed CD49f antagonists and their biological evaluation in breast cancer cells

Abstract Breast cancer is the second cause of cancer death in women and the large majority of those deaths are due to drug resistance and/or recurrence. The cancer stem cell (CSC) model suggests that tumor stem-like cells play key roles in resistance and recurrence in breast cancer patients. Thus, CSCs have been pointed as targets for new anti-cancer therapies. CD49f is an integrin subunit that participates in the CSC-niche interaction. Knock-down of CD49f in breast cancer cells impairs mammosphere formation in vitro and tumorigenesis in vivo, suggesting that this protein is needed for maintenance of stemness. Aiming to target breast CSC, we used consensus docking to select potential CD49f antagonists from a collection of 13,000+ drugs with previous clinical evaluations. Seven compounds with the lowest consensus Z score were selected for in vitro biological validation. Cell adhesion assays showed that four of the selected drugs (5193, 1382, 7631, and 12723) decrease the binding of CD49f+ MDA-MB-231 breast cancer cells to laminin, suggesting that those compounds antagonize the receptor. Mammosphere formation assays showed that compounds 5193, 1382, and 12723 limit the clonogenic capability of CD49f+ breast cancer cells in vitro. Compound 5193 was highly toxic in 2D cell viability assays. On the other hand, the clonogenic impairment produced by compounds 1382 and 12723 is independent of bulk cell line cytotoxicity, suggesting a direct impact on the CSC pool. The analyses of the effect of these drugs on the expression of breast CSC markers and CD49f-activated pathways are on their way. Thus, we have identified some drugs that might be repurposed to target breast CSC. Since the pharmacokinetics and toxicology of those drugs is known, they could easily be ready for clinical trials. We demonstrated that in silico screening is useful to identify antagonists of receptors with relevant roles in breast CSC biology. This work was supported by CONACYT 221105, PAPIIT-UNAM IN228616, and Red temática de células troncales y medicina regenerativa. Citation Format: Velasco-Velázquez M, Velázquez-Quesada I, Aguirre-Alvarado C, Guerrero-Rodríguez S, Ruiz-Moreno A, Ramirez-Salinas G, Segura-Cabrera A, Pérez-Tapia M. Selection of presumed CD49f antagonists and their biological evaluation in breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-06-09.

Breast cancer stem cells expressing different stem cell markers exhibit distinct biological characteristics.

Identification and isolation of breast cancer stem cells (CSCs) based on CD44/CD24 expression and/or enzymatic activity of aldehyde dehydrogenase 1 (ALDH1). However, the differences among the CD44+/CD24−/low cells, ALDH1+ cells and the overlap between the sub-populations have not been frequently investigated. Thus, it is imperative to improve the understanding of breast CSC with different stem markers. CD44+/CD24−/low, ALDH1+ and ALDH1+CD44+/CD24−/low cell populations were isolated from fresh breast cancer tissues and analyzed by flow cytometry and immunofluorescence. Mammosphere formation, cell proliferation assay and Transwell experiments, were used to analyze self-renewal, proliferation and invasion, respectively, for each sub-population. Finally, in vivo experimentation in mice was performed to evaluate the tumorigenic abilities of the sub-populations. The sub-populations of CD44+/CD24−/low, ALDH1+ and ALDH1+CD44+/CD24−/low in human breast cancer cells, represented the 7.2, 4.6 and 1.5% of the total tumor cell population, respectively. ALDH1+CD44+/CD24−/low cells had the strongest ability of self-renewal, invasion, proliferation and tumorigenicity compared with the other sub-populations (P<0.05). In conclusion, different phenotypes of CD44+/CD24−/low, ALDH1+ and ALDH1+CD44+/CD24−/low were isolated and demonstrated that breast CSCs are heterogeneous, and they exhibit distinct biological characteristics. As ALDH1+CD44+/CD24−/low cells demonstrated the strongest stem-like properties, it may be a useful specific stem cell marker. The utilization of more reliable biomarkers to distinguish the breast CSC pool will be important for the development of specific target therapies for breast cancer.

P53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/β-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/β-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNβcat mice displaying a constitutive activation of Wnt/β-catenin signaling in basal cells. K5ΔNβcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNβcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and β-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.

RANK Signaling Blockade Reduces Breast Cancer Recurrence by Inducing Tumor Cell Differentiation.

RANK expression is associated with poor prognosis in breast cancer even though its therapeutic potential remains unknown. RANKL and its receptor RANK are downstream effectors of the progesterone signaling pathway. However, RANK expression is enriched in hormone receptor negative adenocarcinomas, suggesting additional roles for RANK signaling beyond its hormone-dependent function. Here, to explore the role of RANK signaling once tumors have developed, we use the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT), which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacologic approaches demonstrate that therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, decreases tumor and metastasis initiation, and enhances sensitivity to chemotherapy. Mechanistically, genome-wide expression analyses show that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. Moreover, RANK signaling in tumor cells negatively regulates the expression of Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1-population, enriched in tumor-initiating cells. In addition, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with relapse-free tumors. These results support the use of RANKL inhibitors to reduce recurrence and metastasis in breast cancer patients based on its ability to induce tumor cell differentiation. Cancer Res; 76(19); 5857-69. ©2016 AACR.

JARID1B Enables Transit between Distinct States of the Stem-like Cell Population in Oral Cancers.

The degree of heterogeneity among cancer stem cells (CSC) remains ill-defined and may hinder effective anti-CSC therapy. Evaluation of oral cancers for such heterogeneity identified two compartments within the CSC pool. One compartment was detected using a reporter for expression of the H3K4me3 demethylase JARID1B to isolate a JARID1B(high) fraction of cells with stem cell-like function. JARID1B(high) cells expressed oral CSC markers including CD44 and ALDH1 and showed increased PI3K pathway activation. They were distinguished from a fraction in a G0-like cell-cycle state characterized by low reactive oxygen species and suppressed PI3K/AKT signaling. G0-like cells lacked conventional CSC markers but were primed to acquire stem cell-like function by upregulating JARID1B, which directly mediated transition to a state expressing known oral CSC markers. The transition was regulated by PI3K signals acting upstream of JARID1B expression, resulting in PI3K inhibition depleting JARID1B(high) cells but expanding the G0-like subset. These findings define a novel developmental relationship between two cell phenotypes that may jointly contribute to CSC maintenance. Expansion of the G0-like subset during targeted depletion of JARID1B(high) cells implicates it as a candidate therapeutic target within the oral CSC pool. Cancer Res; 76(18); 5538-49. ©2016 AACR.

Numb-like (NumbL) downregulation increases tumorigenicity, cancer stem cell-like properties and resistance to chemotherapy.

NumbL, or Numb-like, is a close homologue of Numb, and is part of an evolutionary conserved protein family implicated in some important cellular processes. Numb is a protein involved in cell development, in cell adhesion and migration, in asymmetric cell division, and in targeting proteins for endocytosis and ubiquitination. NumbL exhibits some overlapping functions with Numb, but its role in tumorigenesis is not fully known. Here we showed that the downregulation of NumbL alone is sufficient to increase NICD nuclear translocation and induce Notch pathway activation. Furthermore, NumbL downregulation increases epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-related gene transcripts and CSC-like phenotypes, including an increase in the CSC-like pool. These data suggest that NumbL can act independently as a tumor suppressor gene. Furthermore, an absence of NumbL induces chemoresistance in tumor cells. An analysis of human tumors indicates that NumbL is downregulated in a variable percentage of human tumors, with lower levels of this gene correlated with worse prognosis in colon, breast and lung tumors. Therefore, NumbL can act as an independent tumor suppressor inhibiting the Notch pathway and regulating the cancer stem cell pool.

Fascin Is Critical for the Maintenance of Breast Cancer Stem Cell Pool Predominantly via the Activation of the Notch Self-Renewal Pathway.

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as "cancer stem cells" (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44hi /CD24lo and ALDH+ ) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799-2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o.

Abstract 5236: Bitter melon juice causes strong efficacy towards pancreatic cancer stem cells by modulating the hedgehog signaling pathway

Abstract According to ACS estimates for year 2015, ∼48,960 new cases of pancreatic cancer (PanC) would be diagnosed with 40,560 associated deaths in the US alone. The median life of PanC patients post diagnosis is &amp;lt;6 months and overall 5 year survival is 3-5%. Thus, there is an urgent need for newer strategies to improve the disease outcome in these patients. Recently, considerable efforts have been directed towards natural dietary/non-dietary agents for both prevention/intervention of PanC. One such dietary agent, bitter melon (Momordica charantia) from Cucurbitaceae family, is now being extensively evaluated for its efficacy against various malignancies including PanC. Recently, we have shown significant growth inhibitory effects of bitter melon juice (BMJ) against a panel of PanC cells including Mia PaCa-2 xenograft tumors. Our studies also showed BMJ activity against both gemcitabine (frontline chemotherapeutic in PanC patients) sensitive as well as resistant PanC cell lines. Since the presence of cancer stem cells (CSC) is now recognized as the main cause for initiation, promotion and drug-resistance as well as relapse of most of the cancers, including PanC, there is a possibility that BMJ was affecting the CSC population in the PanC cells. Accordingly, the present study was aimed to determine whether BMJ has the potential to target CSC in PanC. We isolated CSC enriched (CD44+CD24+EpCAMhigh) cell population from the PanC cell lines, MiaPaCa2, PANC1 and AsPC1 and subjected them as well as the unsorted cells to sphere cluster formation assays in the presence or absence (single vs. multiple time treatment) of BMJ (0.25-2% v/v). The% of floating spheroids generated in the sphere cluster assays after 1-2 weeks were determined. The results indicated a dose-dependent effect of BMJ on both number and size (volume) of spheres, with 2% BMJ showing a significant reduction in both number and size of generated spheres in all PanC cell lines; single treatment of BMJ was found to be equally effective as multiple dosing regimens. Since formation of spheroids under specific in vitro conditions is a measure of stemness, our results show that BMJ has the potential to target the self-renewal of CSCs in PanC cell lines. Subsequent studies employing real time RT-PCR and immunofluorescence showed BMJ effect to be mediated in part by a significant down regulation in the expression of CSC marker CD44, CD133, and hedgehog (Hh)-pathway members in PanC cell lines. Further studies found that BMJ also decreases the protein levels of Hh-pathway members SHH and SMO in MiaPaCa2 xenograft tumors. Together, these are significant findings as they clearly show that BMJ interferes with the kinetics of CSC pool expansion via targeting various regulatory signals associated with the survival and multiplication of CSC pool leading to effective anti-cancer efficacy against PanC cells and their resistant phenotypes. Citation Format: Rajesh Agarwal, Deepanshi Dhar, Chapla Agarwal, Gagan Deep. Bitter melon juice causes strong efficacy towards pancreatic cancer stem cells by modulating the hedgehog signaling pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5236.

Abstract 3325: Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance

Abstract In recent years, impressive technical advancements have been made in the isolation and validation of the mammary stem cells (MaSCs) and cancer stem cells (CSCs). However, the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to resistance to chemotherapy and radiation therapy. However, an effective therapeutic strategy to overcome this resistance is yet to be identified. We have recently discovered that the DNA methyltransferases, especially DNMT1, play critical role in MaSCs and CSCs self-renewal and targeted deletion of this gene impaired mammary tumor formation by inhibiting CSCs formation. However, the molecular mechanism(s) by which DNMTs control CSCs and the therapeutic relevance of DNMTs inhibitors in regulation of CSCs and overcome drug resistance are also largely unknown. In this study, using MMTV-Neu-Tg mouse mammary tumor model, we found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which leads to activation of un-activated Neu-Tg into transformed tumor forming phenotype. Combination of 5-Azacytidine, a DNMT inhibitor, and butyrate, a HDAC inhibitor, markedly reduces CSCs and consequently increases the overall survival of the animal. RNA-seq analysis of the CSCs treated with 5-AzaC+butyrate provides evidence that combined inhibition of DNMTs and HDACs reduces CSCs pool in the mammary gland by blocking growth-promoting signaling molecules like RAD51AP1 and SPC25. RAD51AP1 and SPC25, which are known to play a key role in DNA damage repair and kinetochore assembly, are significantly overexpressed in breast tumor tissues and associated with decreased overall patients’ survival. Further, these two genes are overexpressed in Tamoxifen and Taxol resistant human breast cancer cell lines. Functional inactivation of these genes in breast caner cells facilitates chemotherapy-induced apoptosis and reduces tumor growth. Overall, our studies provide strong evidence that breast CSCs (both basal stem cells and luminal progenitor cells) are susceptible for genetic and epigenetic modifications and associated with resistance to chemo- and radiotherapy. Thus, combination of DNMT and HDAC inhibitors can serve as an effective therapeutic strategy to block mammary tumor growth and to overcome drug resistance by inhibiting CSCs. Citation Format: Rajneesh Pathania, Ravindra B. Kolhe, Sabarish Ramachandran, Gurusamy Mariappan, Priyanka Thakur, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3325.

Antibiotics inhibit sphere-forming ability in suspension culture.

BackgroundThis last decade, a lot of emphasis has been placed on developing new cancer cell culture models, closer to in vivo condition, in order to test new drugs and therapies. In the case of colorectal cancer, the use of patient biopsies to seed 3D primary cultures and mimic tumor initiation necessitates the use of antibiotics to prevent microbial intestinal contamination. However, not only long term use of antibiotics may mask the presence of low levels of microbial contamination, it may also impact cancer cell phenotype.MethodsIn this study we tested the impact of penicillin-streptomycin cocktail addition in both monolayer and suspension culture. To ensure the reliability of our observations we used six different cell lines and each experiment was performed in triplicate. Results were analyzed with Student’s t test.ResultsWe show that penicillin–streptomycin cocktail inhibits the sphere-forming ability of six cancer cell lines in suspension culture though it has no impact in monolayer culture. We correlate this effect with a significant decrease of cancer stem cells pool which holds self-renewal potential.ConclusionsOverall, this study warns against systematic addition of antibiotics in growth medium and raises the interesting possibility of using antibiotics to target cancer stem cells.

Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyses of Functionally Enriched Stem and Progenitor Pools

SummaryThe identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in breast cancer. Estrogen receptor (ER)α+ tumors featured a clear hierarchical organization with switch-like and gradual transitions between different clusters, illustrating how breast cancer cells transfer between discrete differentiation states in a sequential manner. ERα− breast cancer showed less prominent clustering but shared a quiescent cancer stem cell pool with ERα+ cancer. The cellular organization model was supported by single-cell data from primary tumors. The findings allow us to understand the organization of breast cancers at the single-cell level, thereby permitting better identification and targeting of cancer stem cells.