Induction Of Cancer Stem Cells Research Articles

Abstract 2505: GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers

Abstract Despite advancement in cancer therapy, the ability of cancer cells to evade therapy often leads to tumor recurrence and distant metastasis, and ultimately to the death of the patient. Increasing evidence shows that these properties of recurrence can be attributed to a small population of cells within the tumor termed cancer stem cells (CSCs). Understanding the mechanisms that support the growth of CSCs could provide new tools to target these cells and improve therapeutic outcomes. G12 proteins, comprised of Gα12 (GNA12) and Gα13 (GNA13) are mediators of G protein coupled receptor (GPCR) signaling. These proteins are highly expressed in several cancers, and blocking their signaling can significantly inhibit cancer cell invasion and metastasis. Recently, we have shown that loss of GNA13 alone can suppress invasion of breast and prostate cancer cells. Moreover, we have recently obtained evidence that enhanced expression of GNA13 is capable of inducing a stem cell phenotype in cancer cells. Specifically, enforced expression of GNA13 in cancer cells containing low levels of GNA13 protein significantly induced CSC population as shown by increased aldehyde dehydrogenase (ALDH1) activity, increased CD44+/CD24− cells, and enhancement of tumor sphere formation. In contrast, in cancer cells that express high levels of GNA13, knockdown of GNA13 suppressed ALDH1 activity, the CD44+/CD24− cell population, and the formation of spheres. Using reporter assays we have identified that GNA13 induced CSC phenotype most probably via activating Jun Kinase - AP1 signaling pathway and blocking this pathway could significantly inhibit CSC phenotype induced by GNA13. Most importantly we have identified that GNA13 expressing cells are resistant to most commonly used chemotherapeutic agents and ionizing radiations. Blocking GNA13 using shRNAs sensitized these cells to both chemo- and radiotherapy significantly. We therefore hypothesize that GNA13 plays a pivotal role in the induction of CSCs and drug resistance, and the suppression of GNA13 activity may provide therapeutic advantage to cancer therapy. Citation Format: Hui Sun Leong, Suhail Ahmed Kabber Rasheed, Manikandan Lakshmanan, Anandh Kumar Raju, Vinay Tergaonkar, Daniel shao-Weng Tan, Patrick Casey, N.Gopalakrishna Iyer. GNA13 induction of stemness and resistance to chemotherapy in breast and head and neck cancers. [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 2505.

Abstract 3315: Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target

Abstract Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH and WNT up-regulation. EP4 antagonist (EP4A) treatment ablated SLCs both in vitro and in vivo. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 human breast cancer cell lines (named MCF-7-COX-2 and SKBR-3-COX-2) resulted in aggressive phenotypes: increased migration/invasion/proliferation, EMT, elevated SLCs, evidenced by spheroid formation for successive generations, increased ALDH activity and co-expression of COX-2/SLC markers. These changes were reversed with COX-2 inhibitor or EP4A, indicating dependence on COX-2/EP4 activities. COX-2 overexpression or EP4 agonist treatment of COX-2 low cells caused up-regulation of NOTCH/WNT pathway genes, blocked with PI3K/AKT inhibitors. Supporting above findings, micro-array analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes in MCF-7-COX-2 cells. MCF-7-COX-2 cells showed increased orthotopic tumorigenicity and spontaneous multi-organ metastases in NOD/SCID/IL-2Rγ-deficient mice for successive generations with limiting cell inocula. Orthotopic tumors showed significant up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4 knockdown or EP4A treatment. COX-2, EP4 and ALDH1A expression in situ in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. High COX-2/EP4 expression was linked with poor survival. Thus EP4 represents a novel SLC-ablative target in human breast cancer. (Supported by a grant of the OICR to PKL and a TBCRU fellowship to MM) Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Elena Tutunea-Fatan, Mauricio Rodriguez-Torres, Krista Vincent, Andrew Deweyert, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target. [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 3315.

Cisplatin to induce cancer stem cell state in ovarian cancer.

e17098Background: Ovarian cancer (OC) is the most lethal gynecological cancer. Despite high rates of response to initial cytoreductive surgery and taxane-platinum combination therapy, vast majority...

Snail controls proliferation of Drosophila ovarian epithelial follicle stem cells, independently of E-cadherin

Epithelial stem cells undergo constant self-renewal and differentiation to maintain the homeostasis of epithelial tissues that undergo rapid turnover. Recent studies have shown that the epithelial-mesenchymal transition (EMT), which is primarily mediated by Snail via the suppression of E-cadherin, is able to generate cells with stem cell properties. However, the role of Snail in epithelial stem cells remains unclear. Here, we report that Snail directly controls proliferation of follicle stem cells (FSCs) in Drosophila females. Disruption of Snail expression in FSCs compromises their proliferation, but not their maintenance. Conversely, FSCs with excessive Snail expression display increased proliferation and lifespan, which is accompanied by a moderate decrease in the expression of E-cadherin (required for adhesion of FSCs to their niche) at the junction between their adjacent cells, indicating a conserved role of Snail in E-cadherin inhibition, which promote epithelial cell proliferation. Interestingly, a decrease in E-cadherin in snail-knock down FSCs does not restore the decreased proliferation of snail-knock down FSCs, suggesting that adhesion strength of FSCs to their niche is dispensable for Snail-mediated FSC division. Our results demonstrate that Snail controls epithelial stem cell division independently of its known role in the EMT, which contributes to induction of cancer stem cells.

A convenient and effective strategy for the enrichment of tumor-initiating cell properties in prostate cancer cells.

Stem-like prostate cancer (PrCa) cells, also called PrCa stem cells (PrCSCs) or PrCa tumor-initiating cells (PrTICs), are considered to be involved in the mediation of tumor metastasis and may be responsible for the poor prognosis of PrCa patients. Currently, the methods for PrTIC sorting are mainly based on cell surface marker or side population (SP). However, the rarity of these sorted cells limits the investigation of the molecular mechanisms and therapeutic strategies targeting PrTICs. For PrTIC enrichment, we induced cancer stem cell (CSC) properties in PrCa cells by transducing three defined factors (OCT3/4, SOX2, and KLF4), followed by culture with conventional serum-containing medium. The CSC properties in the transduced cells were evaluated by proliferation, cell cycle, SP assay, drug sensitivity technology, in vivo tumorigenicity, and molecular marker analysis of PrCSCs compared with parental cells and spheroids. After culture with serum-containing medium for 8days, the PrCa cells transduced with the three factors showed significantly enhanced CSC properties in terms of marker gene expression, sphere formation, chemoresistance to docetaxel, and tumorigenicity. The percentage of CD133+/CD44+ cells was ninefold higher in the transduced cell population than in the adherent PC3 cell population (2.25 ± 0.62 vs. 0.25 ± 0.12%, respectively), and the SP increased to 1.22 ± 0.18% in the transduced cell population, but was undetectable in the adherent population. This method can be used to obtain abundant PrTIC material and enables a complete understanding of PrTIC biology and development of novel therapeutic agents targeting PrTICs.

Aspirin Suppresses the Acquisition of Chemoresistance in Breast Cancer by Disrupting an NFκB-IL6 Signaling Axis Responsible for the Generation of Cancer Stem Cells.

Acquired chemoresistance has curtailed cancer survival since the dawn of chemotherapy. Accumulating evidence suggests a major role for cancer stem cells (CSC) in chemoresistance, although their involvement in acquired resistance is still unknown. The use of aspirin has been associated with reduced cancer risk and recurrence, suggesting that the anti-inflammatory drug may exert effects on CSCs. In this study, we investigated the contribution of CSCs to acquired chemoresistance of breast cancer and the avenues for reversing such effects with aspirin. We observed that the residual risk of recurrence was higher in breast cancer patients who had acquired chemoresistance. Treatment of preexisting CSCs with a genotoxic drug combination (5-fluorouracil, doxorubicin, and cyclophosphamide) generated an NFκB-IL6-dependent inflammatory environment that imparted stemness to nonstem cancer cells, induced multidrug resistance, and enhanced the migration potential of CSCs. Treatment with aspirin prior to chemotherapy suppressed the acquisition of chemoresistance by perturbing the nuclear translocation of NFκB in preexisting CSCs. Therefore, disruptions to the NFκB-IL6 feedback loop prevented CSC induction and sensitized preexisting CSCs to chemotherapy. Collectively, our findings suggest that combining aspirin and conventional chemotherapy may offer a new treatment strategy to improve recurrence-free survival of breast cancer patients. Cancer Res; 76(7); 2000-12. ©2016 AACR.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24− cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

Iron induces cancer stem cells and aggressive phenotypes in human lung cancer cells.

Evidence has accumulated in support of the critical impact of cancer stem cells (CSCs) behind the chemotherapeutic failure, cancer metastasis, and subsequent disease recurrence and relapse, but knowledge of how CSCs are regulated is still limited. Redox status of the cells has been shown to dramatically influence cell signaling and CSC-like aggressive behaviors. Here, we investigated how subtoxic concentrations of iron, which have been found to specifically induce cellular hydroxyl radical, affected CSC-like subpopulations of human non-small cell lung carcinoma (NSCLC). We reveal for the first time that subchronic iron exposure and higher levels of hydroxyl radical correlated well with increased CSC-like phenotypes. The iron-exposed NSCLC H460 and H292 cells exhibited a remarkable increase in propensities to form CSC spheroids and to proliferate, migrate, and invade in parallel with an increase in level of a well-known CSC marker, ABCG2. We further observed that such phenotypic changes induced by iron were not related to an epithelial-to-mesenchymal transition (EMT). Instead, the sex-determining region Y (SRY)-box 9 protein (SOX9) was substantially linked to iron treatment and hydroxyl radical level. Using gene manipulations, including ectopic SOX9 overexpression and SOX9 short hairpin RNA knockdown, we have verified that SOX9 is responsible for CSC enrichment mediated by iron. These findings indicate a novel role of iron via hydroxyl radical in CSC regulation and its importance in aggressive cancer behaviors and likely metastasis through SOX9 upregulation.

Oncogenic role of leptin and Notch interleukin-1 leptin crosstalk outcome in cancer.

Obesity is a global pandemic characterized by high levels of body fat (adiposity) and derived-cytokines (i.e., leptin). Research shows that adiposity and leptin provide insight on the link between obesity and cancer progression. Leptin's main function is to regulate energy balance. However, obese individuals routinely develop leptin resistance, which is the consequence of the breakdown in the signaling mechanism controlling satiety resulting in the accumulation of leptin. Therefore, leptin levels are often chronically elevated in human obesity. Elevated leptin levels are related to higher incidence, increased progression and poor prognosis of several human cancers. In addition to adipose tissue, cancer cells can also secrete leptin and overexpress leptin receptors. Leptin is known to act as a mitogen, inflammatory and pro-angiogenic factor that induces cancer cell proliferation and tumor angiogenesis. Moreover, leptin signaling induces cancer stem cells, which are involved in cancer recurrence and drug resistance. A novel and complex signaling crosstalk between leptin, Notch and interleukin-1 (IL-1) [Notch, IL-1 and leptin crosstalk outcome (NILCO)] seems to be an important driver of leptin-induced oncogenic actions. Leptin and NILCO signaling mediate the activation of cancer stem cells that can affect drug resistance. Thus, leptin and NILCO signaling are key links between obesity and cancer progression. This review presents updated data suggesting that adiposity affects cancer incidence, progression, and response to treatment. Here we show data supporting the oncogenic role of leptin in breast, endometrial, and pancreatic cancers.

Stemming Colorectal Cancer Growth and Metastasis: HOXA5 Forces Cancer Stem Cells to Differentiate

Wnt signaling drives colorectal cancer stem cells, but effective therapeutics targeting these cells and their signaling pathways are lacking. In this issue of Cancer Cell, Ordóñez-Morán and colleagues describe a promising therapeutic intervention for colorectal cancers that selectively induces cancer stem cell differentiation through HOXA5 expression and Wnt signaling inhibition.

EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC.

Endothelial cells induce cancer stem cell features in differentiated glioblastoma cells via bFGF.

BackgroundGlioblastoma multiforme (GBM) is a rapidly growing malignant brain tumor, which has been reported to be organized in a hierarchical fashion with cancer stem cells (CSCs) at the apex. Recent studies demonstrate that this hierarchy does not follow a one-way route but can be reverted with more differentiated cells giving rise to cells possessing CSC features. We investigated the role of tumor microvascular endothelial cells (tMVECs) in reverting differentiated glioblastoma cells to CSC-like cells.MethodsWe made use of primary GBM lines and tMVECs. To ensure differentiation, CSC-enriched cultures were forced into differentiation using several stimuli and cultures consisting solely of differentiated cells were obtained by sorting on the oligodendrocyte marker O4. Reversion to the CSC state was assessed phenotypically by CSC marker expression and functionally by evaluating clonogenic and multilineage differentiation potential.ResultsConditioned medium of tMVECs was able to replenish the CSC pool by phenotypically and functionally reverting differentiated GBM cells to the CSC state. Basic fibroblast growth factor (bFGF), secreted by tMVECs, recapitulated the effects of the conditioned medium in inducing re-expression of CSC markers and increasing neurosphere formation ability of differentiated GBM cells.ConclusionsOur findings demonstrate that the CSC-based hierarchy displays a high level of plasticity showing that differentiated GBM cells can acquire CSC features when placed in the right environment. These results point to the need to intersect the elaborate network of tMVECs and GBM CSCs for efficient elimination of GBM CSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0420-3) contains supplementary material, which is available to authorized users.

Prostaglandin E2 Promotes Colorectal Cancer Stem Cell Expansion and Metastasis in Mice

Inflammation may contribute to the formation, maintenance, and expansion of cancer stem cells (CSCs), which have the capacity for self-renewal, differentiation, and resistance to cytotoxic agents. We investigated the effects of the inflammatory mediator prostaglandin E2 (PGE2) on colorectal CSC development and metastasis in mice and the correlation between levels of PGE2 and CSC markers in human colorectal cancer (CRC) specimens. Colorectal carcinoma specimens and matched normal tissues were collected from patients at the Mayo Clinic (Scottsdale, AZ) and analyzed by mass spectrometry and quantitative polymerase chain reaction. Human primary CRC cells and mouse tumor cells were isolated using microbeads orflow cytometry and analyzed for sphere-formation and by flow cytometry assays. LS-174T cells were sorted by flow cytometry (for CD133(+)CD44(+) and CD133(-)CD44(-) cells) and also used in these assays. NOD-scidIL-2Rγ(-/-) (NSG) mice were given cecal or subcutaneous injections of LS-174T or human primary CRC cells. Apc(Min/+) mice and NSG mice with orthotopic cecal tumors were given vehicle (controls), PGE2, celecoxib, and/or Ono-AE3-208. PGE2 downstream signaling pathways were knocked down with small hairpin RNAs, expressed from lentiviral vectors in LS-174T cells, or blocked with inhibitors in human primary CRC cells. Levels of PGE2 correlated with colonic CSC markers (CD133, CD44, LRG5, and SOX2 messenger RNAs) in human colorectal carcinoma samples. Administration of PGE2 to Apc(Min/+) mice increased tumor stem cells and tumor burden, compared with controls. NSG mice given PGE2 had increased numbers of cecal CSCs and liver metastases compared with controls after intracecal injection of LS-174T or human primary CRC cells. Alternatively, celecoxib, an inhibitor of prostaglandin-endoperoxide synthase 2, reduced polyp numbers in Apc(Min/+) mice, liver metastasis in NSG mice with orthotopic tumors, and numbers of CSCs in Apc(Min/+) and NSG mice. Inhibitors or knockdown of PGE2 receptor 4 (EP4), phosphoinositide 3-kinase (PI3K) p85α, extracellular signal-regulated kinase 1 (ERK1), or nuclear factor (NF)-κB reduced PGE2-induced sphere formation and expansion of LS-174T and/or human primary CRC cells. Knockdown of ERK1 or PI3K p85α also attenuated PGE2-induced activation of NF-κB in LS-174T cells. An EP4 antagonist reduced the ability of PGE2 to induce CSC expansion in orthotopic tumors and to accelerate the formation of liver metastases. Knockdown experiments showed that NF-κB was required for PGE2 induction of CSCs and metastasis in mice. PGE2 induces CSC expansion by activating NF-κB, via EP4-PI3K and EP4-mitogen-activated protein kinase signaling, and promotes the formation of liver metastases in mice. The PGE2 signaling pathway therefore might be targeted therapeutically to slow CSC expansion and colorectal cancer progression.

Abstract 1416: Granulocyte-colony stimulating factor (G-CSF) enhances stemness by inducing myeloid-derived suppressor cell (MDSC) in cervical cancer

Abstract Objectives: Granulocyte-colony stimulating factor (G-CSF) is known to induce the myeloid-derived suppressor cell (MDSC) to stimulate the progression of uterine cervical cancer. The aim of the current study is to investigate the role of MDSC in the induction of cancer stem cells in uterine cervical cancer. Methods: We first established cervical cancer cell lines stably transfected with G-CSF expression vector (ME180-GCSF) or control vector (ME180-control). Then, using BALB/c nude mice subcutaneously inoculated with ME180-G-CSF or ME180-control cells, the effect of G-CSF on the induction of MDSCs (CD11b+Gr1+ cells) and cancer stem cells (ALDH-high cells) were assessed by flow cytometry. Using MDSCs isolated from the spleens of ME180-GCSF-bearing mice, we next investigated whether the MDSCs induce cancer stem cells in vitro. Finally, we analyzed the association between the G-CSF expression and ALDH1A1 expression in human cervical cancer specimens by immunohistochemistry. Results: 1) G-CSF induces MDSCs from mice bone marrow. 2) ME180-GCSF-derived tumors contain greater number of MDSCs and ALDH-high cells. 3) Co-incubation of ME180 with MDSCs cells increased the number of ALDH-high cells. 4) A positive correlation was observed between the tumor G-CSF expression and ALDH1A1 expression in human cervical cancers. Conclusions: G-CSF-induced MDSCs increase the number of cancer stem cells in uterine cervical cancer. Citation Format: Hiromasa Kuroda. Granulocyte-colony stimulating factor (G-CSF) enhances stemness by inducing myeloid-derived suppressor cell (MDSC) in cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2015-1416

Abstract 1078: TGF-β and TNF-α activate gene transcription programs associated with poor breast cancer prognosis

Abstract Background: Tumor Growth Factor-β (TGF-β) and Tumor Necrosis Factor-α (TNF-α) are cytokines, which induce the local inflammation and contribute to tumor invasion and metastasis. Recent reports show that TGF-β and TNF-α induce epithelial-to-mesenchymal transition (EMT) in both non-cancer and cancer cells. EMT is a cellular program, through which cancer cells transform from epithelial to mesenchymal phenotype. It is known that EMT induces cancer stem cell (CSC), which is a main culprit in tumor progression. However, it is not well understand what molecular signature drives EMT and CSC induction in the downstream of TGF-β and TNF-α. We hypothesize that TGF-β and TNF-α promote tumor progression and contribute to poor cancer prognosis. The objective of this study is to identify the downstream gene signature of TGF-β and TNF-α stimulation, which allows us to predict poor cancer prognosis. Methods and Results: We previously established in vitro EMT model, in which we stimulated ARPE-19 retinal epithelial cells with TGF-β and TNF-α and observed cellular focuses and fibronectin buildups, hallmarks of in vitro EMT (J Bio Chem 2010). We analyzed microarray transcription profiles of EMT progression at different time points (0, 1, 6, 16, 24, 48, 60 hours). To quantify the likeliness of EMT and CSC, we analyzed EMT and CSC gene scores of the EMT progression by correlation analysis (PNAS 2009). We observed that EMT gene score reached the highest at 16 hours and followed by elevations of CSC gene score, suggesting a dynamic change of gene signature that drives the progression from triggering EMT to inducing CSC by TGF-β and TNF-α. In order to determine which time point of EMT timeframe is more representative for poor cancer prognosis, the transcriptional profile at each time point was compared with the one at 0 hour using t-test and the differentially expressed genes (p<0.05, fold change> 2) were used for further analysis. The expression pattern of each time point was compared with each cancer patient from two breast cancer data sets (Mainz and Transbig) and was used to predict patient prognosis. We discovered that the gene signature at 6-hour (early phase of EMT) was the only predictable signature for the poor prognosis of primary breast cancer patients (logrank p-value of 0.045), with a 5-year distance metastasis free (DMF) survival rate of 0.77 vs. 0.94, and a hazard ratio of 1.49. The same gene signature also predicts poor prognosis in ER-positive primary breast cancer patients (p-value = 0.028, hazard ratio = 1.74). We further performed Ingenuity Pathway Analysis using 6-hour gene signature and observed that interferon response and NF-κ pathway activation were the top ranked pathways. Conclusion: Using time-course transcriptional profiles stimulated by TGF-β and TNF-α, we discovered gene signature that precedes EMT and CSC induction, represented by NF-κ pathway. This signature was significant in predicting prognosis of breast cancer patients. Citation Format: Yuan Qi, Kazuharu Kai, Chad J. Creighton, Bedrich Eckhardt, Hideyuki Saya, Debu Tripathy, Naoto T. Ueno. TGF-β and TNF-α activate gene transcription programs associated with poor breast cancer prognosis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2015-1078

EMT-induced metabolite signature identifies poor clinical outcome.

Metabolic reprogramming is a hallmark of cancer. Epithelial-mesenchymal transition (EMT) induces cancer stem cell (CSC) characteristics and promotes tumor invasiveness; however relatively little is known about the metabolic reprogramming in EMT. Here we show that breast epithelial cells undergo metabolic reprogramming following EMT. Relative to control, cell lines expressing EMT transcription factors show ≥1.5-fold accumulation of glutamine, glutamate, beta-alanine and glycylleucine as well as ≥1.5-fold reduction of phosphoenolpyruvate, urate, and deoxycarnitine. Moreover, these metabolic alterations were found to be predictive of overall survival (hazard ratio = 2.3 (95% confidence interval: 1.31-4.2), logrank p-value = 0.03) and define breast cancer molecular subtypes. EMT-associated metabolites are primarily composed of anapleurotic precursors, suggesting that cells undergoing EMT have a shift in energy production. In summary, we describe a unique panel of metabolites associated with EMT and demonstrate that these metabolites have the potential for predicting clinical and biological characteristics associated with patient survival.

Abstract 5191: Missense mutants of p53 tumor suppressor contributes to drug-resistance and epithelial-mesenchymal transition in colon cancer cells

Abstract The mutants of p53 tumor suppressor that have been detected in approximately 50% of tumors exhibit oncogenic function and promote tumor progression. Restoration of tumor suppression by introduction of wild-type p53 effectively eliminate cancers in preclinical studies; however, more than 80% of p53 mutations are missense mutants, those can form heterotetramers and diminish the therapeutic effect of p53 transgene. It is still unclear how missense mutants promote tumor progression, particularly in whether or not these mutants contribute to induced cancer stem cells during chemotherapy. We report herewith that R273 missense mutant contributes to drug-induced stem cell-like behavior in colorectal cancer cells. Heterozygous SW48 TP53 (R273H/+) cells presented the same sensitivity to doxorubicin and paclitaxel as its parental SW48 colon cancer cells. However, after exposure to lower concentrations of doxorubicin (10 nM) or paclitaxel (4 nM) for eight weeks, SW48 TP53 not only were resistant to anticancer drugs, but also displayed an epithelial-mesenchymal transition (EMT). With the overexpressed E-cadherin and down-expressed vimentin, SW48 TP53 cells that were exposed to doxorubicin displayed significantly EMT morphological changes and increased wound healing, compared to SW 48 cells. Interestingly, inhibition of ceramide glycosylation with PDMP to increase endogenous ceramide restored wild-type p53 expression in heterozygous SW48 TP53 cells exposed to doxorubicin and further sensitized these cells to anticancer drugs and reversed EMT. These findings indicate that a heterozygous missense p53 mutant promotes EMT and drug resistance of colon cancer cells in chemotherapy, and restoration of p53 expression with suppression ceramide glycosylation can improve therapeutic efficacy. Citation Format: Salman B. Hosain, Yong Y. Liu. Missense mutants of p53 tumor suppressor contributes to drug-resistance and epithelial-mesenchymal transition in colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2015-5191

Abstract 2245: SIPA1 negatively regulates the survive in breast cancer patients and promotes CD44 expression to drive cancer cell stemness

Abstract Background: SIPA1 (signal-induced proliferation-associated protein 1) encodes for a GTPase-activating protein, known to be a negative regulator of Ras-related Protein (RAP1). It has been implicated in cancer invasion and metastasis, including cervical cancer, breast cancer and colorectal cancer. In our previous study of SIPA1 activates the integrin beta1 to promote metastasis, we found that SIPA1 may effect series of surface adhesive protein expression, including an important glyco-protein, CD44. CD44 is reported as a critical marker of breast cancer stem cells, so we hypothesis that SIPA1 may induce the stemness in breast cancer through regulating the expression of CD44. Methods: We examined the relationship between the expression of SIPA1 and CD44 in breast cancer tissues and different breast cancer cell lines. Then we used the knockdown of SIPA1 in MDA-MB-231 and an overexpression in the MDA-MB-361 to examine the effects of SIPA1 expression to CD44 in breast cancer cell lines. In vitro tumorigenic properties of SIPA1 high cells were characterized with sensitivity to 5FU and the ability to form tumorspheres, the expression of cancer stem cell markers, including CD44, CD24, ALDH, and the proportion. In vivo analysis used a tissue microarray chip with clinicopathologic features and survival data from 240 breast cancer patients were reviewed with immunohistochemistry (IHC) against SIPA1. Results: In vitro studies showed that SIPA1 high cells with a high level of CD44 expression. They displayed an obviously EMT phenotype, and found to have a higher ability to form tumor spheres, improved expression level of breast cancer stem cell markers, increased resistance to 5FU, and decreased in the cell proliferation. SIPA1 may induce cancer stem cell features through the expression of CD44 and induced the EMT through CD44-PI3K-Akt pathway with a changed CD44 splicing isoform pattern. Studies with breast cancer tissues showed that SIPA1 expressed in the in situ breast cancer, while SIPA1 and CD44 are co-expressed in the metastasis breast cancer. The Kaplane-Meier survival analysis of 240 breast cancer samples found that SIPA1 negative-associated with breast cancer survive (p = 0.003) and prognosis (p = 0.002), and the Cox's proportianal hazard model analysis of prognostic factors in patients with potentially curatively treated breast cancer showed that SIPA1 expression correlated with 5 disease free survive(p = 0.047) and 5 year overall survive(p = 0.015). Conclusion: Taking together, our data suggest that SIPA1 induced CD44 expression in breast cancer cells, SIPA1/CD44high cells display stemness and EMT phenotype, and found to be more resistance to 5FU with a decreased proliferation ability. SIPA1 expression contributes to the survival and prognosis of breast cancer. Further studies to investigate how SIPA1 promotes CD44 expression are needed. Citation Format: Li Su, Jing Xia. SIPA1 negatively regulates the survive in breast cancer patients and promotes CD44 expression to drive cancer cell stemness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2245. doi:10.1158/1538-7445.AM2015-2245

Abstract 1384: CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of triple-negative breast cancer

Abstract Antiangiogenic agents are designed to impede the development of new tumor blood vessels, thereby starving tumors of oxygen and nutrients to ultimately block tumor growth. Despite advances made in developing antiangiogenic therapies, clinical and preclinical data suggest that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenic agents and patients frequently fail to demonstrate significantly better overall survival. Acquired resistance to antiangiogenic agents has been attributed in part to the induction of intra-tumoral hypoxia and the concomitant stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. In this preclinical model, we demonstrate that CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (CPT), can reverse the stimulatory effects of hypoxia on the CSC population in an orthotopic triple-negative xenograft model. The payload, camptothecin, a topoisomerase 1 inhibitor, inhibits HIF-1α protein accumulation at concentrations below those that were cytotoxic to tumor cells, and inhibits hypoxia-mediated CSC induction at these low concentrations in vitro. In an orthotopic triple-negative breast cancer model, CRLX101 is synergistic with bevacizumab. Tumor reimplantation experiments confirm that the combination therapy effectively targets both the bulk tumor cell and CSC populations. Results generated from these preclinical studies support the clinical research of combining antiangiogenic agents with CRLX101 to increase patient survival. CRLX101 is currently in phase 2 clinical trials in combination with bevacizumab in both recurrent ovarian cancer and metastatic renal cell carcinoma. Citation Format: Sarah J. Conley, Trenton L. Baker, Joseph P. Burnet, Rebecca L. Thiesen, Douglas Lazarus, Christian G. Peters, Shawn G. Clouthier, Scott Eliasof, Max S. Wicha. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1384. doi:10.1158/1538-7445.AM2015-1384

Heat shock factor 1 induces cancer stem cell phenotype in breast cancer cell lines.

Heat shock factor 1 (HSF1) has long been recognized as the master transcription factor that regulates heat shock proteins (HSPs). More recently HSF1 has been associated with a broader role in regulating response to a variety of cellular stresses beyond heat-shock. We previously found that high HSF1 expression is associated with poor outcome in lung, breast and colon cancers. Importantly, however, the HSF1 signature correlated with poor outcome in these studies was not related to the heat shock response, which suggested that tumor outcome associated with high HSF expression may be due to processes other than stress response. Hence, we explored the question whether high HSF1 expression might be associated with the cancer stem cell (CSC) phenotype. To do so, we examined the association of HSF1 with CSC phenotype by FACS and immunofluorescence. In addition, we evaluated the effects of HSF1 over-expression and knock-down on sphere formation and CSC marker expression in breast cancer cell lines. Here, we report results demonstrating that high HSF1 not only correlates with CSC marker expression, but inducible HSF1 over-expression augments and HSF1 knock-down inhibits CSC phenotype. Furthermore, HSF1 expression confers resistance to chemotherapeutic drugs and increases CSC frequency. In conclusion, our study indicates that one of the potential HSP-independent HSF1 driven mechanisms that may contribute to poor outcome in human tumors involves regulation of the CSC phenotype. Hence, therapeutic inhibition of HSF1 may be one route to target CSCs in human tumors.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-015-3521-1) contains supplementary material, which is available to authorized users.