Increased ALDH Activity Research Articles

P65/RelA NF-κB fragments generated by RIPK3 activity regulate tumorigenicity, cell metabolism, and stemness characteristics.

Receptor‐interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces caspase‐mediated p65/RelA cleavage, resulting in N‐terminal 1‐361 and C‐terminal 362‐549 fragments. We show here that a noncleavable p65/RelA D361E mutant expressed in DA1‐3b leukemia cells decreases mouse survival times and that coexpression of p65/RelA fragments increases the tumorigenicity of B16F1 melanoma cells. This aggressiveness in vivo did not correlate with NF‐κB activity measured in vitro. The fragments and p65/RelA D361E mutant induced different expression profiles in DA1‐3b and B16F1 cells. Stemness markers were affected: p65/RelA D361E increased ALDH activity in DA1‐3b cells, and fragment expression increased melanoma sphere formation in B16/F1 cells. p65/RelA fragments and the D361E noncleavable mutant decreased oxidative or glycolytic cell metabolism, with differences observed between models. Thus, p65/RelA cleavage initiated by kinase‐independent RIPK3 activity in cancer cells is not neutral and induces pleiotropic effects in vitro and in vivo that may vary across tumor types.

Radiation-Induced Bystander Effects of Adipose-Derived Mesenchymal Stem Cells.

ObjectiveThe interaction of tumor cells with surrounding stem cells such as adipose-derived mesenchymal stem cells (ASCs) would be a crucial mechanism of tumor progression. It has been shown that irradiation can affect tumor microenvironment through different mechanisms. Given that, we aimed to examine the bystander radiation-induced effects of ASCs on different cancer cell lines.Materials and MethodsIn this experimental study, ASCs were extracted from five healthy donors, cultured and then irradiated with a 5Gy of gamma radiation. Following 72 hours of incubation, irradiated ASCs-conditioned media (IACM) and non-irradiated ASCs-conditioned media (NIACM) were collected. Following incubation of different cell lines, Jurkat, LNCaP, U87-MG, MDA-MB-231 and MCF-7, in different media, DMEM, NIACM, and IACM, ALDEFLUOR assay and wound healing assays, were conducted. Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression of ATP-binding cassette transporter genes, ABCA1 and ABCG2, was measured in these cell lines. ResultsNIACM significantly increased ALDH activity in MDA-MB-231 cell (P=0.02), while IACM was associated with significant decrease in the LNCaP and MCF-7 cell lines, respectively P=0.02, P=0.03, compared to DMEM as the control. The area of the scratch site was significantly reduced in MDA-MB-231 cells cultured with NIACM compared to DMEM (P=0.04). Furthermore, ABCA1 mRNA expression was considerably decreased in IACM- but not in DMEMtreated LNCaP line (P=0.01).ConclusionIt seems, after exposing to radiation, ASCs modify to prevent tumor development and metastasis through their radiation-induced bystander effects. Therefore, a better understanding of ASCs function in the tumor microenvironment may provide new insights into therapeutic strategies to surmount radio-resistance in cancer treatment.

Elevated PDK1 Expression Drives PI3K/AKT/MTOR Signaling Promotes Radiation-Resistant and Dedifferentiated Phenotype of Hepatocellular Carcinoma.

Resistance to radiotherapy (IR), with consequent disease recurrence, continues to limit the efficacy of contemporary anticancer treatment for patients with hepatocellular carcinoma (HCC), especially in late stage. Despite accruing evidence implicating the PI3K/AKT signaling pathway in cancer-promoting hypoxia, cancerous cell proliferation and radiotherapy-resistance, it remains unclear which molecular constituent of the pathway facilitates adaptation of aggressive HCC cells to tumoral stress signals and drives their evasion of repeated IR-toxicity. This present study investigated the role of PDK1 signaling in IR-resistance, enhanced DNA damage repair and post-IR relapse, characteristic of aggressive HCC cells, while exploring potential PDK1-targetability to improve radiosensitivity. The study employed bioinformatics analyses of gene expression profile and functional protein–protein interaction, generation of IR-resistant clones, flow cytometry-based ALDH activity and side-population (SP) characterization, siRNA-mediated loss-of-PDK1function, western-blotting, immunohistochemistry and functional assays including cell viability, migration, invasion, clonogenicity and tumorsphere formation assays. We showed that the aberrantly expressed PDK1 characterizes poorly differentiated HCC CVCL_7955, Mahlavu, SK-HEP1 and Hep3B cells, compared to the well-differentiated Huh7 or normal adult liver epithelial THLE-2 cells, and independently activates the PI3K/AKT/mTOR signaling. Molecular ablation of PDK1 function enhanced susceptibility of HCC cells to IR and was associated with deactivated PI3K/AKT/mTOR signaling. Additionally, PDK1-driven IR-resistance positively correlated with activated PI3K signaling, enhanced HCC cell motility and invasiveness, augmented EMT, upregulated stemness markers ALDH1A1, PROM1, SOX2, KLF4 and POU5F1, increased tumorsphere-formation efficiency and suppressed biomarkers of DNA damage—RAD50, MSH3, MLH3 and ERCC2. Furthermore, the acquired IR-resistant phenotype of Huh7 cells was strongly associated with significantly increased ALDH activity, SP-enrichment, and direct ALDH1-PDK1 interaction. Moreover, BX795-mediated pharmacological inhibition of PDK1 synergistically enhances the radiosensitivity of erstwhile resistant cells, increased Bax/Bcl-2 apoptotic ratio, while suppressing oncogenicity and clonogenicity. We provide preclinical evidence implicating PDK1 as an active driver of IR-resistance by activation of the PI3K/AKT/mTOR signaling, up-modulation of cancer stemness signaling and suppression of DNA damage, thus, projecting PDK1-targeting as a putative enhancer of radiosensitivity and a potential new therapeutic approach for patients with IR-resistant HCC.

Radiation induces an inflammatory response that results in STAT3-dependent changes in cellular plasticity and radioresistance of breast cancer stem-like cells

Purpose: Pro-inflammatory cytokines within the tumor microenvironment, such as IL-6, contribute to the maintenance of stem cells and promote their survival following treatment. The IL-6/STAT3 pathway is a key regulator of genes involved in cancer progression. Activation of STAT3 promotes expansion of cancer stem cells in triple negative breast cancer. Radiation has also been shown to expand cancer stem cell populations and can induce stemness in nonstem cells. However, the role of IL-6/STAT3 in radiation-induced changes in cellular plasticity is unclear.Materials and methods: Expression and secretion of IL-6 from triple-negative breast cancer cell lines SUM159PT and MDA-MB-231 were determined after radiation treatment by real-time PCR and ELISA. Activation of STAT3 after radiation was determined by western blotting. Changes in cellular plasticity induced by radiation were determined by examining ALDEFLUOR activity, gene expression analysis of aldehyde dehydrogenase isoforms and mammosphere forming assays with and without the addition of STAT3 inhibitors. To determine the effect of radiation on nonstem cell populations, experiments were also carried out in ALDEFLUOR sorted cells.Results: Radiation induced an inflammatory response in both cell lines that resulted in activation of STAT3. Additionally, radiation induced a stem-like state as evidenced by an increased activity and expression of the ALDH isoforms ALDH1A1 and ALDH1A3, and increased self-renewal capabilities. Radiation increased ALDH activity and self-renewal in non-stem cell (ALDH−) populations, suggesting radiation-induced cellular reprograming. However, inhibition of STAT3 blocked the radiation-induced stem-like state in both ALDEFLUOR positive and negative populations, and enhanced radiosensitivity.Conclusions: Radiation-induced changes in cellular plasticity are STAT3 dependent and may be a potential target to reduce radioresistance in TNBC and improve treatment outcome.

Disulfiram Overcomes Cisplatin Resistance in Human Embryonal Carcinoma Cells.

Cisplatin resistance in testicular germ cell tumors (TGCTs) is a clinical challenge. We investigated the underlying mechanisms associated with cancer stem cell (CSC) markers and modalities circumventing the chemoresistance. Chemoresistant models (designated as CisR) of human embryonal carcinoma cell lines NTERA-2 and NCCIT were derived and characterized using flow cytometry, gene expression, functional and protein arrays. Tumorigenicity was determined on immunodeficient mouse model. Disulfiram was used to examine chemosensitization of resistant cells. ALDH1A3 isoform expression was evaluated by immunohistochemistry in 216 patients’ tissue samples. Chemoresistant cells were significantly more resistant to cisplatin, carboplatin and oxaliplatin compared to parental cells. NTERA-2 CisR cells exhibited altered morphology and increased tumorigenicity. High ALDH1A3 expression and increased ALDH activity were detected in both refractory cell lines. Disulfiram in combination with cisplatin showed synergy for NTERA-2 CisR and NCCIT CisR cells and inhibited growth of NTERA-2 CisR xenografts. Significantly higher ALDH1A3 expression was detected in TGCTs patients’ tissue samples compared to normal testicular tissue. We characterized novel clinically relevant model of chemoresistant TGCTs, for the first time identified the ALDH1A3 as a therapeutic target in TGCTs and more importantly, showed that disulfiram represents a viable treatment option for refractory TGCTs.

Regulation of Osteosclerosis by Inoculated Cd133+ PC3 Cells in Bone-marrow Microenvironmental Niches.

ABSTRACTBone is the most common site of prostate cancer (PC) metastasis. Studies suggest that cancer stem cells (CSCs) are associated with stemness characteristics, providing some support for the concept that CSCs act as osteosclerotic precursors in bone microenvironmental niches. Here, we asked whether ectopic overexpression of CD133 maintains stability of CSCs in human PC cell lines and induces the changes of molecular features in the bone microenvironment. Ectopic overexpression of CD133 in PC3 or DU145 cells led to increased expression of ALDHA1, OCT4, and NANOG, enhanced colony‐forming ability, and increased ALDH activity. In addition, micro‐CT imaging, confocal microscopy, and H&E staining of mouse tissue confirmed that CD133 overexpression in PC3 and DU145 led to marked osteolytic bone tumor. However, expression of osteoblastic markers such as collagen type I, bone sialoprotein, and osteocalcin (OC) at the tumor margin of CD133‐overexpressing PC3 tumors in mouse tibiae was higher than that of CD133‐overexpressing DU145 tumors with osteosclerotic molecular features. In addition, expression of osteopontin (OPN) mRNA/protein by CD133‐overexpressing PC3 cells was higher than that by DU145 cells. Especially, conditioned medium (CM) from PC3CD133+ cells increased osterix (OSX) activity in bone marrow stromal cells (BMSCs), resulting in increased expression of OC mRNA/protein resulted in increased staining of mineralized matrix by Alizarin red. However, CM from OPN silenced PC3CD133+ cells led to a reduction of OC mRNA and protein expression through OSX activity resulted in reduced amount of mineralized matrix. In conclusion, these findings suggest that CD133 plays a functional role in regulating CSC characteristics in PCs and modulates their abilities in which induce the osteosclerosis of BMSCs. In addition, OPN from CSCs acts as a niche component that promotes osteosclerosis by supporting osteoblastic differentiation of BMSCs. © 2019 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

High metabolic rate and stem cell characteristics of esophageal cancer stem-like cells depend on the Hsp27-AKT-HK2 pathway.

Tumor progression with chemoresistance and local recurrence is commonly happened during treatment of esophageal squamous cell carcinoma (ESCC). Cancer stem cells (CSC) may respond for tumor progression. However, there are few reports regarding metabolism of esophageal CSCs with clinical correlation. In this work, we demonstrated that ESCC cell lines in spheroid culture display CSC phenotypes, including increased ALDH activity, chemoresistance and tumor initiation, which are dependent on Hsp27 activation. Esophageal CSCs also exhibit reprogrammed metabolic features particularly higher glycolysis and oxidative phosphorylation, which are regulated via the Hsp27-AKT-HK2 pathway. Moreover, HK2 is required for maintenance of CSC phenotypes. Inhibition of CSC metabolism reduces cell growth and tumor formation. Clinically, patients who underwent surgical resection for esophageal cancer, and displayed overexpression of both Hsp27 and HK2, had the worst prognosis of all expression types. In conclusion, stem cells features and aberrant metabolic reprogramming of esophageal CSCs depend on the Hsp27-AKT-HK2 pathway. Targeting Hsp27 and HK2 could be novel therapeutic strategy for treating esophageal cancer and warrants further investigation.

MEK5/ERK5 activation regulates colon cancer stem-like cell properties

Colon cancer has been proposed to be sustained by a small subpopulation of stem-like cells with unique properties allowing them to survive conventional therapies and drive tumor recurrence. Identification of targetable signaling pathways contributing to malignant stem-like cell maintenance may therefore translate into new therapeutic strategies to overcome drug resistance. Here we demonstrated that MEK5/ERK5 signaling activation is associated with stem-like malignant phenotypes. Conversely, using a panel of cell line-derived three-dimensional models, we showed that ERK5 inhibition markedly suppresses the molecular and functional features of colon cancer stem-like cells. Particularly, pharmacological inhibition of ERK5 using XMD8-92 reduced the rate of primary and secondary sphere formation, the expression of pluripotency transcription factors SOX2, NANOG, and OCT4, and the proportion of tumor cells with increased ALDH activity. Notably, this was further associated with increased sensitivity to 5-fluorouracil-based chemotherapy. Mechanistically, ERK5 inhibition resulted in decreased IL-8 expression and NF-κB transcriptional activity, suggesting a possible ERK5/NF-κB/IL-8 signaling axis regulating stem-like cell malignancy. Taken together, our results provide proof of principle that ERK5-targeted inhibition may be a promising therapeutic approach to eliminate drug-resistant cancer stem-like cells and improve colon cancer treatment.

CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer

High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. We hypothesize that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, we observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.

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.

COX-2 Induces Breast Cancer Stem Cells via EP4/PI3K/AKT/NOTCH/WNT Axis.

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/WNT signaling. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 cell lines resulted in: increased migration/invasion/proliferation, epithelial-mesenchymal transition (EMT), elevated SLCs (spheroid formation), increased ALDH activity and colocalization of COX-2 and SLC markers (ALDH1A, CD44, β-Catenin, NANOG, OCT3/4, SOX-2) in spheroids. These changes were reversed with COX-2-inhibitor or EP4-antagonist (EP4A), indicating dependence on COX-2/EP4 activities. COX-2 over-expression or EP4-agonist treatments of COX-2-low cells caused up-regulation of NOTCH/WNT genes, blocked with PI3K/AKT inhibitors. NOTCH/WNT inhibitors also blocked COX-2/EP4 induced SLC induction. Microarray analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes. MCF-7-COX-2 cells showed increased mammary tumorigenicity and spontaneous multiorgan metastases in NOD/SCID/IL-2Rγ-null mice for successive generations with limiting cell inocula. These tumors showed 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 of the MCF-7-COX-2 cells. COX-2/EP4/ALDH1A mRNA expression in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. In situ immunostaining of human breast tumor tissues revealed co-localization of SLC markers with COX-2, supporting COX-2 inducing SLCs. High COX-2/EP4 mRNA expression was linked with reduced survival. Thus, EP4 represents a novel SLC-ablative target in human breast cancer. Stem Cells 2016;34:2290-2305.

Low adherent cancer cell subpopulations are enriched in tumorigenic and metastatic epithelial-to-mesenchymal transition-induced cancer stem-like cells.

Cancer stem cells are responsible for tumor progression, metastasis, therapy resistance and cancer recurrence, doing their identification and isolation of special relevance. Here we show that low adherent breast and colon cancer cells subpopulations have stem-like properties. Our results demonstrate that trypsin-sensitive (TS) breast and colon cancer cells subpopulations show increased ALDH activity, higher ability to exclude Hoechst 33342, enlarged proportion of cells with a cancer stem-like cell phenotype and are enriched in sphere- and colony-forming cells in vitro. Further studies in MDA-MB-231 breast cancer cells reveal that TS subpopulation expresses higher levels of SLUG, SNAIL, VIMENTIN and N-CADHERIN while show a lack of expression of E-CADHERIN and CLAUDIN, being this profile characteristic of the epithelial-to-mesenchymal transition (EMT). The TS subpopulation shows CXCL10, BMI-1 and OCT4 upregulation, differing also in the expression of several miRNAs involved in EMT and/or cell self-renewal such as miR-34a-5p, miR-34c-5p, miR-21-5p, miR-93-5p and miR-100-5p. Furthermore, in vivo studies in immunocompromised mice demonstrate that MDA-MB-231 TS cells form more and bigger xenograft tumors with shorter latency and have higher metastatic potential. In conclusion, this work presents a new, non-aggressive, easy, inexpensive and reproducible methodology to isolate prospectively cancer stem-like cells for subsequent biological and preclinical studies.

Abstract 3905: Stem like cells in human breast cancer: EP4 as a therapeutic target

Abstract Background: A tumor cell subset known as stem like cells (SLC) defy conventional therapies, requiring identification of SLC-specific therapeutic targets. We established that elevated COX-2 expression by breast cancer cells promotes tumor progression via inactivation of host immune cells, enhancement of cancer cell migration and invasiveness, and promotion of tumor-associated angiogenesis and lymphangiogenesis primarily by activation of the PGE-2 receptor EP4 on tumor and host cells. Cardiovascular side effects of long term use of COX-2 inhibitors argued for an alternative target, potentially EP4. Objectives/approaches: We asked whether COX-2 or EP4 additionally stimulates SLC in breast cancer. COX-2 and HER-2, often co-expressed in human breast cancer, are both major determinants of cancer progression. To define the roles of COX-2 in HER2 +ve and -ve cells, COX-2 cDNA was stably transfected into COX-2-low, HER-2-ve, ER+ve, non-metastatic MCF-7 and COX-2-ve, ER-ve, HER-2-high SKBR-3 human breast cancer cell lines to derive MCF-7-COX-2 and SKBR-3-COX-2 cells which were tested for changes in phenotype and functions in vitro and in vivo. Results: Both COX-2 over-expressing lines showed (1) EMT (low E-Cadherin and high Vimentin, Twist and N-Cadherin), (2) higher proliferation/ migration/ invasion, (3) upregulation of EP4 and angiogenic/ lymphangiogenic factors VEGF-A/C/D, (4) markedly increased SLC contents indicated by clonogenic spheroid formation for successive generations in vitro, increased ALDH activity and co-expression of COX-2 and multiple SLC markers in spheroids. (5) COX-2 expression markedly increased during spheroid formation in COX-2 disparate cell lines, indicating a selection of COX-2 expressing cells. (6) EP4 activation of COX-2 low cell lines with PGE2 or EP4 agonists markedly increased spheroid-forming efficiency. (7) Functional changes listed above (2-5) were blocked with COX-2 inhibitors or an EP4 antagonist (EP4A), indicating dependence on COX-2 and EP4 activity. (8) MCF-7-COX-2 cells showed dramatically increased lung colony forming ability and growth at 4-6 wks in NOD/SCID/GUSB null mice (identified by GUSB staining), which were blocked with EP4A treatment or EP4 knockdown; (9) MCF-7-COX-2 (as low as 5000) cells showed increased orthotopic tumorigenicity in NOD/SCID/IL-2Rγ deficient mice on transplantation for successive generations. (10) Finally, expression of COX-2 and EP4 were positively correlated with the SLC marker ALDH in human breast cancer tissues, and associated with poor patient survival. Conclusion/ Significance: EP4 represents a novel therapeutic target to eradicate SLCs in breast cancer. EP4 antagonists may spare cardiovascular side effects of COX-2 inhibitors, primarily attributed to cardio protective effects of PGI2. (Supported by the CBCF, Ontario Chapter and OICR grants to PKL, TBCRU and CIHR-STP fellowships to MM, CBCF fellowship to EL and a gift of EP4A, RQ-15986 by RaQualia Pharma). Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Gillian Bell, Erin Landman, Mauricio Rodriguez- Torres, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Stem like cells in human breast cancer: EP4 as a therapeutic target. [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 3905. doi:10.1158/1538-7445.AM2014-3905

Abstract A51: Cancer stem cell marker ALDH1A3 is associated with increased resistance of Lovo-1 colon cancer cells to autophosphorylation inhibitor of FAK.

Abstract Introduction: Focal Adhesion Kinase is an important survival signal in cancer. Recently, we demonstrated that the autophosphorylation inhibitor of FAK, Y15 effectively inhibited colon cancer cell viability in vitro and blocked xenograft tumor growth in vivo. Among several colon cancer cell lines, Lovo-1 was more resistant to Y15 than other cell lines. To reveal signaling pathways responsible for increased resistance of Lovo-1 cells to autophosphorylation inhibitor of FAK, we performed microarray gene profile study in SW620 and Lovo-1 cells treated with Y15 inhibitor. Experimental Procedures: MTT assay and clonogenicity assays were used to detect the effect of Y15 on viability and colony formation. Western blotting was used to detect protein expression. IlluminaRef 8 whole genome gene expression microarray, bioinformatics and statistical analyses were performed to detect significantly affected genes by Y15 in two colon cancer cell lines: SW620 and Lovo-1 cells. PicoProbe ALDH activity assay kit (Biovision) was used to detect ALDH activity. ALDH1A3 siRNA was used to inhibit ALDH1A3 expression. The specific ALDH inhibitor, diethylaminobenzaldehyde was used to inhibit ALDH activity. Results: MTT, clonogenicity assays and Western blotting with Y397-FAK antibody demonstrated that Lovo-1 colon cancer cells were more resistant to Y15 treatment than SW620 colon cancer cells. The microarray analysis detected 12 up- and 4 significantly down-regulated genes that were affected >2-fold (p<0.05) in Lovo-1, but not in SW620 cells. Among unique genes up-regulated by Y15 in Lovo-1 cells, a stem cell marker- ALDH1A3 was detected to be up-regulated >2-fold. ALDH activity kit demonstrated high activity of ALDH in Lovo-1 colonospheres, and the spheres were resistant to Y15 treatment. Treatment with ALDH1A3 siRNAs sensitized Lovo-1 cells to Y15 inhibitor and decreased cell viability more effectively than each agent alone. The same result was obtained with ALDH inhibitor, DEAB in combination with Y15 inhibitor. Conclusion: The microarray study revealed several important genes up- or down-regulated in Lovo-1 cancer cells in response to Y15 inhibitor compared to SW620 cells. The Lovo-1 cells formed spheres with increased ALDH activity. A cancer stem cell marker, ALDH1A3 was associated with increased resistance of Lovo-1 cancer cells to Y15. In addition, down-regulation of ALDH1A3 and FAK effectively decreased viability of Lovo-1 colon cancer cells, resistant to FAK inhibitor that can be used as a novel combination therapy approach in resistant cell lines. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A51. Citation Format: Baotran Ho, Melissa Heffler, Jeffrey Conroy, Liu Song, Dan Wang, William G. Cance, Vita M. Golubovskaya. Cancer stem cell marker ALDH1A3 is associated with increased resistance of Lovo-1 colon cancer cells to autophosphorylation inhibitor of FAK. [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 A51.

Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

IntroductionInherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.MethodsAn isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.ResultsCharacterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.ConclusionOur results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.

S6-5: Obesity Drives Epithelial-to-Mesenchymal Transition and Tumor Progression in a Novel Claudin-Low Mammary Cancer Model.

Abstract Background: Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative tumor initiating cell (TIC) population. However, the exact mechanism of obesity-driven tumor progression remains unknown. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. Materials, Methods, and Results: To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice, designated M-Wnt and E-Wnt. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative TIC population (62% +/−7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the TIC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key TIC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). Discussion: In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, which demonstrates that clinical subtypes of breast cancer are differentially regulated by energy balance modulation. Additionally, our data demonstrates, for the first time, that energy balance modulation (ie, CR and obesity) directly regulates EMT, in response to local upregulation of TGF-β signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S6-5.

Abstract 3332: Obesity drives epithelial/mesenchymal transition and tumor progression in a novel Wnt-1 mammary cancer model

Abstract Epidemiological evidence suggests a potential role of obesity in regulating clinical subtype, differentiation status, and prognosis of breast cancer. Specifically, an elevated waist-hip ratio is associated with increased risk and progression of basal-like breast cancer, an aggressive form characterized by heterogeneous tumors typically enriched in a putative breast cancer stem cell (BCSC) population. Therefore, we hypothesized that obesity regulates a plastic population of multipotent malignant cells. To test this hypothesis, we generated and characterized two distinct murine mammary tumor cell lines derived from MMTV-Wnt-1 transgenic mice. M-Wnt cells displayed a mesenchymal morphology while E-Wnt cells had an epithelial morphology. M-Wnt cells harbored a large CD44+/CD24- putative cancer stem cell population (62% +7.8), had significant mammosphere forming capacity (>30% of cells form mammospheres, p<0.0001), and increased ALDH activity (7% are ALDH+, p=0.0004). M-Wnt cells have increased migration (scratch assay) and invasion in vitro (226-fold higher after 30h, p<0.0001). As few as 50 unsorted M-Wnt cells, injected into C57BL/6 mice were capable of forming a tumor. Microarray analysis revealed that M-Wnt cells display gene expression profiles virtually identical to human Claudin-low breast tumors, while E-Wnt cells clustered with basal-like tumors. EMT and stem cell gene expression patterns of M-Wnt cells were maintained in vivo, including decreased E-cadherin and increased N-cadherin, fibronectin, vimentin, SNAIL, TWIST, SLUG, FOXC2, and TGF-β (p<0.05 for all). Using these cell lines in vivo, we tested the hypothesis that energy balance modulation, through diet-induced obesity (DIO) and calorie restriction (CR), regulates the BCSC population. We found that M-Wnt tumors, transplanted into ovariecomized syngeneic female C57BL/6 mice, grew at significantly different growth rates depending on the diet treatment (DIO > Control, p=0.011; CR < Control, p=0.012), while E-Wnt tumors were only affected by CR (CR < Control p=0.001; no difference between DIO and Control tumors). DIO enhanced M-Wnt tumor progression, adipocyte infiltration, and central necrosis and drove EMT (decreased E-Cadherin and increased fibronectin and N-Cadherin expression) through modulation of key BCSC associated genes, including increased TGF-β, SNAIL, FOXC2, and Oct4 (p<0.05 for all). In conclusion, we found that the mesenchymal M-Wnt cell line is highly responsive to changes in dietary energy balance status relative to the E-Wnt differentiated epithelial cell line, suggesting that energy balance modulation (ie, CR and obesity) regulates tumor growth through direct regulation of a BCSC population, possibly in response to alterations in TGF-β signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3332. doi:10.1158/1538-7445.AM2011-3332

Moving Forward in Human Mammary Stem Cell Biology and Breast Cancer Prognostication Using ALDH1

In this issue of Cell Stem Cell, the seminal work of Ginestier et al. (2007) elegantly demonstrates that aldehyde dehydrogenase 1 (ALDH1) facilitates the identification and isolation of normal and malignant human mammary stem cells in vitro, in vivo, and in situ in fixed tissues.