Colon Cancer-initiating Cells Research Articles

Exosomes, metastases, and the miracle of cancer stem cell markers.

Cancer-initiating cells (CIC) are the driving force in tumor progression. There is strong evidence that CIC fulfill this task via exosomes (TEX), which modulate and reprogram stroma, nontransformed cells, and non-CIC. Characterization of CIC, besides others, builds on expression of CIC markers, many of which are known as metastasis-associated molecules. We here discuss that the linkage between CIC/CIC-TEX and metastasis-associated molecules is not fortuitously, but relies on the contribution of these markers to TEX biogenesis including loading and TEX target interactions. In addition, CIC markers contribute to TEX binding- and uptake-promoted activation of signaling cascades, transcription initiation, and translational control. Our point of view will be outlined for pancreas and colon CIC highly expressing CD44v6, Tspan8, EPCAM, claudin7, and LGR5, which distinctly but coordinately contribute to tumor progression. Despite overwhelming progress in unraveling the metastatic cascade and the multiple tasks taken over by CIC-TEX, there remains a considerable gap in linking CIC biomarkers, TEX, and TEX-initiated target modulation with metastasis. We will try to outline possible bridges, which could allow depicting pathways for new and expectedly powerful therapeutic interference with tumor progression.

Abstract B01: Histone acetyltransferase 1 contributes to colon cancer initiating cell chemoresistance through DNA damage repair pathways

Abstract Despite advances in treatment, Colorectal cancer (CRC) remains a leading cause of cancer related deaths in North America. Most CRCs contain a subset of self-renewing colon cancer-initiating cells (CC-ICs) responsible for tumor initiation and maintenance. CC-ICs are relatively chemoresistant as compared to the bulk tumor cells, however the mechanisms of this chemoresistance are poorly understood. An increasing amount of evidence suggests that epigenetic regulators may be playing a central role in driving CC-IC chemoresistance; one such example being histone acetyltransferase (HAT1), which plays an important role in cancer cell proliferation and response to DNA damage (Xue et al, Int J Clin Exp Pathol, 2014). The cytoplasmic HAT1/HAT2 complex acetylates histone H4 on lysine residues 5 and/or 12. Acetylated histone H4 is then bound to histone H3 and the complex is transported to the nucleus where the HAT1/HAT2 complex aids in depositing H3/H4 onto DNA (Parthum, Oncogene, 2007). In normal colon, HAT1 is predominantly localized to the nucleus in cells at the crypt base, whereas in primary and metastatic colorectal tumors, HAT1 expression is upregulated, mostly cytoplasmic, and diffuse throughout the tissue (Seiden-Long et al, Oncogene, 2006). To investigate HAT1 localization in our primary derived CC-IC enriched cell lines we used protein fractionation and western blot analysis. In our samples, we consistently see elevated cytoplasmic HAT1 expression compared to nuclear extracts. To determine the effect of the loss of HAT1 on CC-IC viability we infected our CC-IC enriched cell lines with lentiviral shRNA to knockdown HAT1. We observed no changes in growth or cell viability at baseline between the HAT1 knockdown cells and controls, however, previous studies have shown that HAT1 knockdown increases cancer cell sensitivity to DNA damaging agents such as camptothecin (CPT) or methyl methane sulfonate (MMS) by controlling Rad52 degradation, accumulating replication block double stranded breaks and prolonging G2 arrest, indicating that it is important for DNA repair processing (Barman et al, BBRC, 2006). To determine whether HAT1 contributes to CC-IC chemoresistance we treated our HAT1 knockdown cells with standard of care (SOC) chemotherapy (oxaliplatin). Viability assays showed that after 10 days of treatment there is a 40% decrease in cell viability compared to controls. To investigate whether the decrease in cell viability seen in HAT1 knockdown cells is due to an increase in DNA damage we stained cells for DNA damage marker γH2AX. Immunofluorescence analysis revealed a higher level of γH2AX staining in HAT1 knockdown cells treated with SOC chemotherapy. To examine the effects of HAT1 knockdown on CC-IC function when combined with SOC chemotherapy, we preformed a limiting dilution assay. Untreated CC-IC enriched HAT1 knockdown cells had no significant difference in CC-IC frequency, however, treating these cells with SOC chemotherapy resulted in a decrease in CC-IC frequency in vitro (212.2 vs 52.8). Our current research suggests that HAT1 plays an important role in CC-IC function, chemoresistance and response to DNA damage. We are further investigating HAT1 as a potential therapeutic target in order to improve outcomes for CRC patients. Citation Format: Lauren Agro, Cherry Leung, Yadong Wang, Evelyne Lima-Fernandes, Catherine O'Brien. Histone acetyltransferase 1 contributes to colon cancer initiating cell chemoresistance through DNA damage repair pathways [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B01.

Abstract 1709: STRAP mediates the stemness of human colorectal cancer cells by epigenetic regulation of Notch pathway

Abstract Human colon cancer-initiating cells (CCICs) are responsible for the unrestrained cell proliferation and chemoresistance of colorectal tumors. Notch signaling is critical for the maintenance and self-renewal of cancer-initiating cells in colon cancer. We have shown that the serine-threonine kinase receptor-associated protein (STRAP), a ubiquitous WD40 protein, is involved in promoting tumorigenesis through activated MAPK/ERK pathway and pRb phosphorylation. In addition, our previous studies uncovered that STRAP is up-regulated in 60% colon carcinomas and behaves as an oncogene in a TGF-β-dependent or -independent manner. Based on our new finding that STRAP stabilizes intracellular fragment of Notch3 (ICN3) via post-translational modification, we sought to explore the cross-talk between STRAP and Notch pathway in regulating stemness of CCICs. Here, we have generated STRAP knock down (KD) colorectal cancer cell lines (RKO, DLD1, HCT116, HT29, WiDr, SW480, SW620, and LOVO) to show how STRAP abrogates apoptosis by up-regulating CD44+/CD133+ subpopulations of CCICs. Heterozygous Strap KO inhibits tumorigenesis accompanied by the reduction of stemness phenotype (CD44+, Lgr5+) in a colitis-associated mouse model. Western blot assays and immunofluorescene staining demonstrated that the expression of stemness biomarkers (Nanog, Oct4 and Sox2) is decreased in STRAP KD cells. Furthermore, the transcriptional activation of Notch pathway related genes is downregulated in the STRAP KD cells. EpiTect Chip qPCR Arrays profiled that 17 out of 84 genes key to Notch signal transduction had higher activating (H3K4me3) and lower repressive (H3K27me3) histone modifications in WT cells compared to STRAP KD cells. Among these genes, we identified two transcription factors, Hes1 and Hes5, that are transcriptionally regulated by STRAP as demonstrated by luciferase assays and qPCR. We further demonstrated that Hes1 and Hes5 were transcriptionally activated by STRAP through Polycomb Repressive Complex 2 (PRC2)-dependent mechanism. Immunoprecipitation experiments showed that STRAP interacts with the key components, EZH2 and SUZ12 of PRC2 complex in vivo, which antagonizes the histone methyltransferase activity in H3K27 on the promoters of Hes1 and Hes5, resulting in activating these two genes. Of interest, ectopic expression of either Hes1 or Hes5 can rescue the stemness phenotype in STRAP KD cells. Together, our findings provide a novel mechanistic insight into the regulation of CCICs via STRAP-PRC2-Hes1/5 regulatory axis, which represents a potential target for CCICs, thus overcoming cancer chemoresistance and relapse. Citation Format: Lin Jin, Trung Tam Vu, Pran K. Datta. STRAP mediates the stemness of human colorectal cancer cells by epigenetic regulation of Notch 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 1709.

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model.

BackgroundColorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth.MethodsIn this study we used human HCT-116 colon cancer ALDH+ cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA.ResultsWe observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated β-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206+). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1β, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1β, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2.ConclusionOur data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2466-z) contains supplementary material, which is available to authorized users.

Olfactory Receptor Family 7 Subfamily C Member 1 Is a Novel Marker of Colon Cancer-Initiating Cells and Is a Potent Target of Immunotherapy.

Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy. Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model. OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model. OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; 22(13); 3298-309. ©2016 AACR.

NOTCH Signaling Regulates Asymmetric Cell Fate of Fast- and Slow-Cycling Colon Cancer-Initiating Cells.

Colorectal cancer cells with stem-like properties, referred to as colon cancer-initiating cells (CCIC), have high tumorigenic potential. While CCIC can differentiate to promote cellular heterogeneity, it remains unclear whether CCIC within a tumor contain distinct subpopulations. Here, we describe the co-existence of fast- and slow-cycling CCIC, which can undergo asymmetric division to generate each other, highlighting CCIC plasticity and interconvertibility. Fast-cycling CCIC express markers, such as LGR5 and CD133, rely on MYC for their proliferation, whereas slow-cycling CCIC express markers, such as BMI1 and hTERT, are independent of MYC. NOTCH signaling promotes asymmetric cell fate, regulating the balance between these two populations. Overall, our results illuminate the basis for CCIC heterogeneity and plasticity by defining a direct interconversion mechanism between slow- and fast-cycling CCIC. Cancer Res; 76(11); 3411-21. ©2016 AACR.

Targeting phospholipase D1 attenuates intestinal tumorigenesis by controlling β-catenin signaling in cancer-initiating cells.

Expression of the Wnt target gene phospholipase D1 (PLD1) is up-regulated in various carcinomas, including colorectal cancer (CRC). However, the mechanistic significance of its elevated expression in intestinal tumorigenesis remains unknown. In this study, we show that genetic and pharmacological targeting of PLD1 disrupts spontaneous and colitis-associated intestinal tumorigenesis in Apc(Min/+) and azoxymethane/dextran sodium sulfate mice models. Intestinal epithelial cell-specific PLD1 overexpression in Apc(Min/+) mice accelerated tumorigenesis with increased proliferation and nuclear β-catenin levels compared with Apc(Min/+) mice. Moreover, PLD1 inactivation suppressed the self-renewal capacity of colon cancer-initiating cells (CC-ICs) by decreasing expression of β-catenin via E2F1-induced microRNA (miR)-4496 up-regulation. Ultimately, low expression of PLD1 coupled with a low level of CC-IC markers was predictive of a good prognosis in CRC patients, suggesting in vivo relevance. Collectively, our data reveal that PLD1 has a crucial role in intestinal tumorigenesis via its modulation of the E2F1-miR-4496-β-catenin signaling pathway. Modulation of PLD1 expression and activity represents a promising therapeutic strategy for the treatment of intestinal tumorigenesis.

Proteoglycans as potential microenvironmental biomarkers for colon cancer.

Glycosylation changes occur widely in colon tumours, suggesting glycosylated molecules as potential biomarkers for colon cancer diagnostics. In this study, proteoglycans (PGs) expression levels and their transcriptional patterns are investigated in human colon tumours in vivo and carcinoma cells in vitro. According to RT-PCR analysis, normal and cancer colon tissues expressed a specific set of PGs (syndecan-1, perlecan, decorin, biglycan, versican, NG2/CSPG4, serglycin, lumican, CD44), while the expression of glypican-1, brevican and aggrecan was almost undetectable. Overall transcriptional activity of the PGs in normal and cancer tissues was similar, although expression patterns were different. Expression of decorin and perlecan was down-regulated 2-fold in colon tumours, while biglycan and versican expression was significantly up-regulated (6-fold and 3-fold, respectively). Expression of collagen1A1 was also increased 6-fold in colon tumours. However, conventional HCT-116 colon carcinoma and AG2 colon cancer-initiating cells did not express biglycan and decorin and were versican-positive and -negative, respectively, demonstrating an extracellular origin of the PGs in cancer tissue. Selective expression of heparan sulfate (HS) proteoglycans syndecan-1 and perlecan in the AG2 colon cancer-initiating cell line suggests these PGs as potential biomarkers for cancer stem cells. Overall transcriptional activity of the HS biosynthetic system was similar in normal and cancer tissues, although significant up-regulation of extracellular sulfatases SULF1/2 argues for a possible distortion of HS sulfation patterns in colon tumours. Taken together, the obtained results suggest versican, biglycan, collagen1A1 and SULF1/2 expression as potential microenvironmental biomarkers and/or targets for colon cancer diagnostics and treatment.

The impact of inflammatory lipid mediators on colon cancer-initiating cells.

The role of inflammatory lipid-mediators in tumor progression is well recognized in colorectal cancer; however, if this includes promotion of cancer-initiating cells remains unclear. We show that the inflammatory lipid-mediators leukotriene D4 and prostaglandin E2 increased the Aldehyde dehydrogenase (ALDH(+) ) population, the colony formation capacity, and tumor growth in a xenograft model of colon cancer. The ALDH(+) cells showed significant resistance to irradiation and 5-fluorouracil treatment that could be further augmented by these lipid-mediators, occurring in parallel with increased target gene expression. Our data emphasize a role for tumor microenvironment derived inflammatory lipid-mediators to favor cancer stem cells-like characteristics and thus promote tumor progression.

A metabolic signature of colon cancer initiating cells.

Colon cancer initiating cells (CCICs) are more tumorigenic and metastatic than the majority of colorectal cancer (CRC) cells. CCICs have also been associated with stem cell-like properties. However, there is a lack of system-level understanding of what mechanisms distinguish CCICs from common CRC cells. We compared the transcriptomes of CD133+ CCICs and CD133- CRC cells from multiple sources, which identified a distinct metabolic signature for CD133(high) CCICs. High-resolution unbiased metabolomics was then performed to validate this CCIC metabolic signature. Specifically, levels of enzymes and metabolites involved in glycolysis, the citric acid (TCA) cycle, and cysteine and methionine metabolism are altered in CCICs. Analyses of the alterations further suggest an epigenetic link. This metabolic signature provides mechanistic insights into CCIC phenotypes and may serve as potential biomarkers and therapeutic targets for future CRC treatment.

899: Identifying genes involved in the colon cancer initiating cells (CICs) survivability against montelukast treatment in xenograft model

899: Identifying genes involved in the colon cancer initiating cells (CICs) survivability against montelukast treatment in xenograft model

Cancer-initiating cells derived from human rectal adenocarcinoma tissues carry mesenchymal phenotypes and resist drug therapies

Accumulating evidence indicates that cancer-initiating cells (CICs) are responsible for cancer initiation, relapse, and metastasis. Colorectal carcinoma (CRC) is typically classified into proximal colon, distal colon, and rectal cancer. The gradual changes in CRC molecular features within the bowel may have considerable implications in colon and rectal CICs. Unfortunately, limited information is available on CICs derived from rectal cancer, although colon CICs have been described. Here we identified rectal CICs (R-CICs) that possess differentiation potential in tumors derived from patients with rectal adenocarcinoma. The R-CICs carried both CD44 and CD54 surface markers, while R-CICs and their immediate progenies carried potential epithelial–mesenchymal transition characteristics. These R-CICs generated tumors similar to their tumor of origin when injected into immunodeficient mice, differentiated into rectal epithelial cells in vitro, and were capable of self-renewal both in vitro and in vivo. More importantly, subpopulations of R-CICs resisted both 5-fluorouracil/calcium folinate/oxaliplatin (FolFox) and cetuximab treatment, which are the most common therapeutic regimens used for patients with advanced or metastatic rectal cancer. Thus, the identification, expansion, and properties of R-CICs provide an ideal cellular model to further investigate tumor progression and determine therapeutic resistance in these patients.

CD49f-positive cell population efficiently enriches colon cancer-initiating cells

Cancer stem cells (CSCs) also known as cancer-initiating cells (CICs) show high tumorigenic activity and high chemo- and radiation resistance. It is, therefore, important to identify CSCs reliably to develop novel curative cancer treatments. In this study, we re-evaluated CSC markers of colorectal cancer for their cellular differentiation and tumorigenic activity, with the aim to identify reliable CSC markers. The rates of change in CD44, CD133, CD166, CD24, CD49f and CXCR4 expression during sodium butyrate (NaBT)-induced cell differentiation were assessed in HT29 and Caco2 colon cancer cell lines. Expression levels of target markers were assessed in clinical CRC samples. Tumorigenic activity was assessed on isolated cell fractions identified by multicolor flow cytometric analysis. In the cell differentiation assay, the average percent change was higher in CD44 (-98.2%) and CD49f (-74.4%) compared to CD133 (-17.9%) and CD166 (-49.4%). Expression of CD24 and CXCR4 appeared random in HT29 and Caco2. Expression of CD44, CD49f, CD133 and CD166 was confirmed in all four clinical CRC samples. Limiting dilution assay of CD44- and CD133-expressing cells revealed that only the CD133⁺CD44⁺ population possessed tumorigenic activity. Tumorigenesis was not affected by CD166 expression. Highly tumorigenic cells could be enriched in samples with higher CD49f expression; CD49f⁺ cells showed high tumorigenesis, whereas CD133⁺ and CD44⁺ cells that were negative for CD49f exhibited no tumorigenic activity. Multicolor analysis revealed that CD49f⁺ cells localized in CD44⁺ and CD133⁺ cell fractions. These findings demonstrated that CD49f is an important marker for identifying colorectal CSCs and suggest that the CD49f⁺ cell fraction may be the best candidate for colorectal CSCs.

Abstract 3747: The impact of lipid inflammatory mediators on colon cancer initiating cells.

Abstract Past studies links the lipid inflammatory mediators at the tumor microenvironment to the colorectal cancer, however their impact on the colon cancer initiating cells (CICs) is largely unknown. Accordingly, the present study aimed to identify the possible link between lipid inflammatory mediators and CICs. In lack of robust marker for identifying colon CICs, we initially screen five different colon cancer cell lines (HCT-116, Caco-2, SW-480 SW-620 and HT-29) for their percentage occurrence using well-established stem cell markers i.e. CD133, CD44 and ALDH. The Flow cytometeric sorting of CD133, CD44 and ALDH subpopulations showed large variation in their population size in these cell lines. Further we noted that ALDH is relatively better marker of these to identify CICs. This statement was based on our observation of markedly increased capacity of ALDH+ cells to produce colonies rather than ALDH− cells. We sorted ALDH+/CICs and investigated the effect of lipid inflammatory mediators such as leukotriene D4 (LTD4) or prostaglandin E2 (PGE2) on these cells. It was noted that LTD4 and PGE2 stimulations not only enriched the relative population of ALDH+ cells; it also greatly improved the colony forming capacity of these cells. Moreover, we observed that ALDH+ cells were more resistant to 5-Fluorouracil (5-FU) treatment compared to ALDH− cells. Furthermore the qPCR data revealed that Bcl-2, COX-2, Bcl-3, ALDH1B1, GLI1 and KLF4 genes were largely amplified in ALDH+ cells in presence of LTD4 or PGE2, indicating their probable impact on the stemness pool of these cells. In conclusion our study shows that lipid inflammatory mediators can affect the survival and stemness of CICs. Citation Format: Anita Sjölander, Kishan Bellamkonda, Wondossen Sime. The impact of lipid inflammatory mediators on colon cancer initiating cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3747. doi:10.1158/1538-7445.AM2013-3747

Prominin-1/CD133 Is Involved in the Lineage Decision As Well As a in the Sensitivity to Molecular Therapy in Ph+ Acute Lymphatic Leukemia.

AbstractAbstract 2579Chronic myelogenous leukemia (CML) and Philadephia chromosome-positive acute lymphatic leukemia (Ph+ ALL) is caused by the t(9;22) which fuses the BCR to ABL resulting in a deregulated tyrosine kinase activity. ABL Kinase inhibitiors (AKI) such as imatinib, are effective in the early stage CML, but in advanced stages patients relapse as a result of point mutations within the BCR/ABL. However a certain group of resistant patients do not have point mutations, which can explain the resistance. The mechanisms of resistance remain often unknown. CD133 (PROMININ-1 or PROM-1; in the mouse: Prom-1) is a novel hematopoietic stem/progenitor cell (HSPC) marker, but its biological function is nearly unknown. Its expression is found on various leukemic cells. It has been shown that PROM-1-negative colon cancer-initiating cells were more aggressive than PROM-1-positive cells, suggesting that cancer-initiating cells are in fact PROM-1-negative.We examined the role of PROM-1 in the normal and BCR/ABL-induced malignant haematopoiesis in Prom-1−/− in comparison to Prom+/+ murine HSPCs. Our results suggest that PROM-1 plays an important role in the induction of the BCR/ABL related leukemic phenotype. BCR/ABL induced a significantly higher colony number in Prom−/− as compared to Prom+/+ HSPCs in factor independent CFU-Assays, which did not respond to 1μM Imatinib. In fact in comparison to Prom +/+ HSPCs Prom −/− HSPCs exhibited in presence as well as in absence of BCR/ABL a different response to Imatinib characterized by a significantly increase of immature c-Kit and Sca-1-positive cells. Furthermore in a transduction/transplantation model, BCR/ABL induced a significantly higher rate of ALLs (50%) in the Prom−/− than in Prom+/+ background, where only CML-like diseases were seen. Based on these results we studied the role of PROM-1 for the non-mutational resistance in Ph+ ALL. We investigated the expression of PROM-1 in different non mutational resistance models of Ph+ ALL among the patient derived cell lines (SupB15 and Tom-1) as well as 7 primary long term cultures derived from patient with Ph+ ALL (PDLTCs). These PDLTCs exhibit several grades of resistance against established ABL-kinase inhibitors (AKI). Furthermore there is a cross resistance against other molecular therapy approaches, such as allosteric inhibition by GNF-2 or oligomerization inhibition by competitive peptides. In these cells there was a direct relationship between PROM-1 expression and response to AKIs. In fact sensitivity to AKIs increased with the expression of PROM-1. In these PDLTCs targeting PROM-1 by siRNA reduced and the over expression of PROM-1 increased the response to AKIs. Interestingly, also the only PDLTC harboring the BCR/ABL-T315I was PROM-1 negative. Furthermore the induction of resistance by increasing concentrations of either Imatinib or Nilotinib in the Ph+ ALL line SupB15 was accompanied by a progressive reduction of PROM-1 expression on the surface (CD133).In summary, our data show for the first time the importance of PROM-1/CD133 for the determination of the leukemic phenotype and as a potential marker for resistance to AKIs in Ph+ ALL, which is actually under examination in cohorts of Ph+ ALL patients with non mutational resistance. Disclosures:No relevant conflicts of interest to declare.

Proliferation State and Polo-Like Kinase1 Dependence of Tumorigenic Colon Cancer Cells

Tumor-initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor-initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo-like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer-initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133(+) colon cancer cells leading to complete growth arrest of colon cancer stem cell-derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133(+) cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor-initiating cells. Quiescent CD133(+) cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor-initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer.

ID1 and ID3 Regulate the Self-Renewal Capacity of Human Colon Cancer-Initiating Cells through p21

There is increasing evidence that some cancers are hierarchically organized, sustained by a relatively rare population of cancer-initiating cells (C-ICs). Although the capacity to initiate tumors upon serial transplantation is a hallmark of all C-ICs, little is known about the genes that control this process. Here, we establish that ID1 and ID3 function together to govern colon cancer-initiating cell (CC-IC) self-renewal through cell-cycle restriction driven by the cell-cycle inhibitor p21. Regulation of p21 by ID1 and ID3 is a central mechanism preventing the accumulation of excess DNA damage and subsequent functional exhaustion of CC-ICs. Additionally, silencing of ID1 and ID3 increases sensitivity of CC-ICs to the chemotherapeutic agent oxaliplatin, linking tumor initiation function with chemotherapy resistance.

Abstract 1228: Interleukin 8 (IL8) is a modulator of tumorigenicity in colon cancer initiating cells

Abstract Introduction: At least 20% of patients with ulcerative colitis (UC) will develop colitis associated cancer (CAC) within 30 years of disease onset. Furthermore, patients with UC have a 5 fold greater risk of developing CAC than the incidence of CRC in the general population. Recently, we isolated and propagated colon cancer initiating cells (CCICs) from UC and CRC patients, and showed that these cells generated tumors in mice and propagated as spheres in vitro. IL8 is a pro-inflammatory chemokine with known proliferation and migration properties. Previously, we showed that inhibition of IL8 inhibited the growth of CCIC-derived tumors in vivo. We hypothesize that IL8 modulates several cancer processes and potentiates the progression from colitis to cancer. Methods: Conditioned media from UC and CRC patient sphere cell isolate cultures were used for measurement of secreted IL8. Exogenous IL8 (10 ng/mL) was added to sphere cultures and proliferation was evaluated. In addition, we knocked down IL8 and IL8 receptor (CXCR1) expression in CRC cell line by short hairpin RNA (shRNA) and measured proliferation in vitro and tumor growth in vivo. We then monitored CCICs from UC or CRC patients, either in the presence or absence of IL8, for symmetric vs. asymmetric division via time lapse video. Results: The chemokine array showed an increase in IL8 in both UC and CRC spheres. Spheres treated with IL8 proliferated faster than controls. Sphere numbers increased with increased exposure to IL8 on days 5 and 7. Conditioned media concentrations of IL8 were increased in both CRC (361-2446 pg/mL) and colitic spheres (208-599 pg/mL) when compared to commercial CRC cell lines (HT29-20.8 and SW480-74.6, respectively). In vitro, knockdown of IL8 or CXCR1 decreased proliferation 1.8-fold compared to the non-targeting controls. Similarly, in vivo xenografts of IL8 or CXCR1 knockdown cells showed 80% decreased tumor growth. UC-derived CCICs exposed to exogenous IL8 exhibited an increase in proliferation starting as early as 16 hrs. We also observed that IL8 treated cells showed a higher percentage of symmetric cellular divisions (68%) when compared to untreated cells (42%). Conclusions: Our findings demonstrate that IL8 potentiates proliferation and symmetric cell division, which is thought to be associated with tumorigenicity. This indicates that IL8 may contribute to the progression of colitis-associated cancer through a modulation of tumorigenic processes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1228. doi:1538-7445.AM2012-1228

Abstract 2773: HSP90 inhibitors target KRAS mutant human tumors through degradation of STK33

Abstract Mutations in the KRAS proto-oncogene occur in ∼30% of human cancers and are particularly prevalent in adenocarcinomas of the pancreas, lung, and colon. Previous efforts to develop drugs that directly inhibit the activity of mutant KRAS have not met with success. We have recently shown that cancer cells driven by mutant KRAS require expression of the serine/threonine kinase STK33 for their viability and proliferation, supporting STK33 as a context-dependent therapeutic target. However, specific strategies for interfering with the critical functions of STK33, which remain to be elucidated, are not yet available. Using a mass spectrometry-based screen for STK33 protein interaction partners, we report that the HSP90/CDC37 chaperone complex and BAG2, a member of the BAG family of molecular chaperone regulators, bind to and stabilize STK33 in human cancer cells. Pharmacologic inhibition of HSP90, using structurally divergent small molecules currently in clinical development, induced proteasome-mediated degradation of STK33 in human cancer cells of various tissue origin in vitro and in vivo, and triggered apoptosis preferentially in KRAS mutant cells in an STK33-dependent manner. Furthermore, HSP90 inhibitor treatment impaired sphere formation and viability of primary human colon cancer-initiating cells harboring mutant KRAS. Similar to HSP90 inhibition, short hairpin RNA-mediated suppression of BAG2 depleted STK33, whereas BAG2 overexpression rescued STK33 protein levels in the presence of HSP90 inhibitors, resulting in diminished apoptosis in KRAS mutant cancer cells. These findings (1) provide mechanistic insight into the activity of HSP90 inhibitors in KRAS mutant cancer cells, (2) indicate that the enhanced requirement for STK33 can be exploited to target mutant KRAS-driven tumors, (3) identify STK33 depletion through HSP90 inhibition as a biomarker-guided therapeutic strategy with immediate translational potential, and (4) point to BAG2 as a candidate cancer drug target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2773. doi:1538-7445.AM2012-2773

STAT3 signaling pathway is necessary for cell survival and tumorsphere forming capacity in ALDH+/CD133+ stem cell-like human colon cancer cells

Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existence of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH+/CD133+). The effects of STAT3 inhibition in colon cancer stem-like cells were examined.The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower IC50 in colon cancer stem-like cells. In summary, our results indicate that STAT3 is a novel therapeutic target in colon cancer stem-like cells and inhibition of STAT3 in cancer stem-like cells may offer a potential treatment for colorectal cancer.