Malaney R. O'Connell

Curcumin promotes autophagic survival of a subset of colon cancer stem cells, which are ablated by DCLK1-siRNA

Curcumin is known to induce apoptosis of cancer cells by different mechanisms, but its effects on cancer stem cells (CSC) have been less investigated. Here, we report that curcumin promotes the survival of DCLK1-positive colon CSCs, potentially confounding application of its anticancer properties. At optimal concentrations, curcumin greatly reduced expression levels of stem cell markers (DCLK1/CD44/ALDHA1/Lgr5/Nanog) in three-dimensional spheroid cultures and tumor xenografts derived from colon cancer cells. However, curcumin unexpectedly induced proliferation and autophagic survival of a subset of DCLK1-positive CSCs. Spheroid cultures were disintegrated by curcumin in vitro but regrew within 30 to 40 days of treatment, suggesting a survival benefit from autophagy, permitting long-term persistence of colorectal cancer. Notably, RNA interference-mediated silencing of DCLK1 triggered apoptotic cell death of colon cancer cells in vitro and in vivo, and abolished colorectal cancer survival in response to curcumin; combination of DCLK1-siRNA and curcumin dramatically reversed CSC phenotype, contributing to attenuation of the growth of spheroid cultures and tumor xenografts. Taken together, our findings confirm a role of DCLK1 in colon CSCs and highlight DCLK1 as a target to enhance antitumor properties of curcumin.

Methods for detecting circulating cancer stem cells (CCSCs) as a novel approach for diagnosis of colon cancer relapse/metastasis.

Cancer stem cells (CSCs) are believed to be resistant to currently available therapies and may be responsible for relapse of cancer in patients. Measuring circulating tumor cells (CTCs) in the blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers or relapse of the cancer disease. However, accurate detection of CTCs has remained a problem, as epithelial-cell markers used to date are not always reliable for detecting CTCs, especially during epithelial–mesenchymal transition. As CSCs are required to initiate metastatic tumors, our goal was to optimize and standardize a method for identifying circulating CSCs (CCSCs) in patients, using established CSC markers. Here, we report for the first time the detection of CCSCs in the blood of athymic nude mice, bearing metastatic tumors, and in the blood of patients positive for colonic adenocarcinomas. Using a simple and non-expensive method, we isolated a relatively pure population of CSCs (CD45−/CK19+), free of red blood cells and largely free of contaminating CD45+ white blood cells. Enriched CCSCs from patients with colon adenocarcinomas had a malignant phenotype and co-expressed CSC markers (DCLK1/LGR5) with CD44/Annexin A2. CSCs were not found in the blood of non-cancer patients, free of colonic growths. Enriched CCSCs from colon cancer patients grew primary spheroids, suggesting the presence of tumor-initiating cells in the blood of these patients. In conclusion, we have developed a novel diagnostic assay for detecting CSCs in circulation, which may more accurately predict the risk of relapse or metastatic disease in patients. As CSCs can potentially initiate metastatic growths, patients positive for CCSCs can be treated with inhibitory agents that selectively target CSCs, besides conventional treatments, to reduce the risk of relapse/metastatic disease for improving clinical outcomes.

Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model

The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

CD90+ stromal cells are the major source of IL-6, which supports cancer stem-like cells and inflammation in colorectal cancer

IL‐6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90+ innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL‐6; however, their contribution to the increase in IL‐6 in CRC and to tumor‐promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL‐6 producing CMFs is increased in CRC (C‐CMFs) and they represent the major source of IL‐6 in T2‐T3 CRC tumors. Activity/expression of stem cell markers‐aldehyde dehydrogenase and LGR5‐ was significantly up‐regulated in colon cancer cells (SW480, Caco‐2 or HT29) cultured in the presence of conditioned medium from tumor isolated C‐CMFs in an IL‐6 dependent manner. C‐CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL‐6 dependent manner. Our study suggests that CD90+ fibroblasts/myofibroblasts may be the major source of IL‐6 in T2‐T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL‐6 producing CAFs (a.k.a. C‐CMFs) may provide a useful target for treating or preventing CRCs.

Epigenetic changes and alternate promoter usage by human colon cancers for expressing DCLK1-isoforms: Clinical Implications

DCLK1 specifically marks colon/pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). Down-regulation of DCLK1 results in loss of cancer-stem-cells (CSCs), and inhibits spheroidal/xenograft growths from hCRC-cells. The 5′-promoter of DCLK1-gene is reportedly hypermethylated in hCRCs, resulting in loss of expression of DCLK1-transcripts, originating from 5′(α)-promoter (termed DCLK1-L, in here). However, in mouse colon-tumors, 5′-promoter of DCLK1-gene remains unchanged, and DCLK1-L, originating from 5′(α)-promoter, is expressed. We hypothesized that elevated levels of DCLK1-protein in hCRC-cells, may be transcribed/translated from an alternate-promoter. Several in silico and molecular biology approaches were used to test our hypothesis. We report for the first time that majority of hCRCs express short-transcripts of DCLK1 (termed DCLK1-S, in here) from an alternate β-promoter in IntronV of the gene, while normal-colons mainly express DCLK1-L from 5′(α)-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating (α)-promoter, while activated NF-κBp65 (bound to NF-κB-cis-element), activates (β)-promoter in cancer-cells. DCLK1-S expression was examined in a cohort of 92 CRC patients; high-expressors had significantly worse overall-survival compared to low-expressors. Our novel findings’ regarding usage of alternate (β)-promoter by hCRCs, suggests that DCLK1-S may represent an important target for preventing/inhibiting colon-cancers, and for eliminating colon-CSCs.

A novel antibody against cancer stem cell biomarker, DCLK1-S, is potentially useful for assessing colon cancer risk after screening colonoscopy

DCLK1 expression is critically required for maintaining growth of human colon cancer cells (hCCCs). Human colorectal tumors (CRCs) and hCCCs express a novel short isoform of DCLK1 (DCLK1-S; isoform 2) from β-promoter of hDCLK1 gene, while normal colons express long isoform (DCLK1-L; isoform 1) from 5′(α)-promoter, suggesting that DCLK1-S, and not DCLK1-L, marks cancer stem cells (CSCs). Even though DCLK1-S differs from DCLK1-L by only six amino acids, we succeeded in generating a monospecific DCLK1-S-Antibody (PS41014), which does not cross-react with DCLK1-L, and specifically detects CSCs. Subcellular localization of S/L-isoforms was examined by immune-electron-microscopy (IEM). Surprisingly, besides plasma membrane and cytosolic fractions, S/L also localized to nuclear/mitochondrial fractions, with pronounced localization of S-isoform in the nuclei and mitochondria. Sporadic CRCs develop from adenomas. Screening colonoscopy is used for detection/resection of growths, and morphological/pathological criteria are used for risk assessment and recommendations for follow-up colonoscopy. But, these features are not precise and majority of the patients will never develop cancer. We hypothesized that antibody-based assay(s), which identify CSCs, will significantly improve prognostic value of morphological/pathological criteria. We conducted a pilot retrospective study with PS41014-Ab, by staining archived adenoma specimens from patients who developed (high-risk), or did not develop (low-risk) adenocarcinomas within 10–15 years. PS41014-Ab stained adenomas from initial and follow-up colonoscopies of high-risk patients, at significantly higher levels (three to fivefold) than adenomas from low-risk patients, suggesting that PS41014-Ab could be used as an additional tool for assessing CRC risk. CRC patients, with high DCLK1-S-expressing tumors (by qRT-PCR), were reported to have worse overall survival than low expressers. We now report that DCLK1-S-specific Ab may help to identify high-risk patients at the time of index/screening colonoscopy.

FOXD3 Regulates CSC Marker, DCLK1-S, and Invasive Potential: Prognostic Implications in Colon Cancer

The 5′ (α)-promoter of the human doublecortin-like kinase 1 (DCLK1) gene becomes epigenetically silenced during colon carcinogenesis, resulting in loss of expression of the canonical long(L)-isoform1 (DCLK1-L) in human colon adenocarcinomas (hCRCs). Instead, hCRCs express a short(S)-isoform2 (DCLK1-S) from an alternate (β)-promoter of DCLK1. The current study, examined if the transcriptional activity of the (β)-promoter is suppressed in normal versus cancerous cells. On the basis of in silico and molecular approaches, it was discovered that FOXD3 potently inhibits the transcriptional activity of the (β)-promoter. FOXD3 becomes methylated in human colon cancer cells (hCCC), with loss of FOXD3 expression, allowing expression of the DCLK1(S) variant in hCCCs/hCRCs. Relative levels of FOXD3/DCLK1(S/L) were measured in a cohort of CRC patient specimens (n = 92), in relation to overall survival (OS). Patients expressing high DCLK1(S), with or without low FOXD3, had significantly worse OS compared with patients expressing low DCLK1(S). The relative levels of DCLK1-L did not correlate with OS. In a pilot retrospective study, colon adenomas from high-risk patients (who developed CRCs in <15 years) demonstrated significantly higher staining for DCLK1(S) + significantly lower staining for FOXD3, compared with adenomas from low-risk patients (who remained free of CRCs). Latter results strongly suggest a prognostic value of measuring DCLK1(S)/FOXD3 in adenomas. Overexpression of DCLK1(S), but not DCLK1(L), caused a significant increase in the invasive potential of hCCCs, which may explain worse outcomes for patients with high DCLK1-S–expressing tumors. On the basis of these data, FOXD3 is a potent repressor of DCLK1-S expression in normal cells; loss of FOXD3 in hCCCs/hCRCs allows upregulation of DCLK1-S, imparting a potent invasive potential to the cells. Mol Cancer Res; 15(12); 1678–91. ©2017 AACR.

Inside the USCAP Journals

Amplification of the 12q13–15 chromosomal region involving the murine double minute-2 (MDM2) locus is characteristic of both well-differentiated and dedifferentiated liposarcoma. Dedifferentiated liposarcoma, the more aggressive form, can be graded using the Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC, or “French”) system. The system has three grades—low (1), intermediate (2), and high (3)—based on combined scores for tumor differentiation, mitotic rate, and level of tumor necrosis. The fold amplification of MDM2 is high but variable between cases. Jour and colleagues used fluorescence in situ hybridization to characterize the amplification level of MDM2. The technique is very helpful diagnostically in cases when the biopsy lacks the characteristic lower-grade adipocytic component. Although the grade 3 dedifferentiated liposarcomas behaved more aggressively in terms of local recurrence than lower-grade liposarcomas on multivariate analysis, higher MDM2 amplification levels did not correlate with local recurrence independently. Grading consequences of PTEN loss in prostate biopsies See page 128

Abstract 543: A short isoform of DCLK1, transcribed from an alternate promoter in human colon cancers, represents a novel biomarker and target for diagnostic and treatment purposes

DCLK1 is a specific marker of colon and pancreatic cancers in mice, and is expressed by human colon adenocarcinomas (hCRCs). A sub-set of DCLK1 +ve colon cancer-stem-cells, are resistant to chemopreventive/chemotherapeutic reagents and undergo autophagic survival, resulting in the relapse of colon-cancer disease. However, down-regulation of DCLK1, along with chemopreventive agents, inhibits spheroidal/xenograft growths from hCRC cells, and eliminates both colon-cancer-stem cells and relapse of the disease. We now know that the 5′-promoter of DCLK1 gene is hypermethylated in hCRCs, but not in mouse colon-tumors, resulting in the loss of expression of DCLK1 long-transcripts (DCLK1-L), from the 5′promoter of DCLK1-gene in humans, but not in mice. We hypothesized that elevated levels of DCLK1 protein, detected in hCRCs, are likely transcribed from an alternate-promoter in humans. We used several in silico and molecular biology approaches to test our hypothesis, and report for the first time that hCRCs express short-transcripts of DCLK1 (DCLK1-S) from an alternate promoter in IntronV of the gene, while normal human colons express the long transcript (DCLK1-L) from 5′-promoter. We additionally report an important role of β-catenin and TCF4/LEF binding-sites for activating 5′-promoter, while NF-κBp65 binding to NF-κB cis element, activates the TATA box containing IntronV-promoter in cancer cells. DCLK1-S expression was examined in a cohort of 92 CRC patients, in relation to overall survival and clinicopathological parameters. High expressors had significantly worse overall-survival compared to low expressors, and DCLK1-S expression was found to be an independent prognostic factor. Conclusions. Our novel findings regarding alternate promoter usage by normal colons vs hCRCs suggest that we can develop strategies for specifically targeting DCLK1-S to eliminate colon cancer-stem-cells, while sparing DCLK1-L functions in neurons and normal cells. Our findings further suggest prognostic/diagnostic value of measuring DCLK1-S in CRC patients. Loss of DCLK1-L expression can also be used as a diagnostic marker for indicating the on-set of epigenetic changes associated with colon carcinogenesis in humans, as an early marker. Citation Format: Malaney R. O9Connell, Shubhashish Sarkar, Gurinder Luthra, Yoshinaga Okugawa, Yuji Toiyama, Ajay Goel, Aakash Gajjar, Suimin Qiu, Lawrence Sowers, Pomila Singh. A short isoform of DCLK1, transcribed from an alternate promoter in human colon cancers, represents a novel biomarker and target for diagnostic and treatment purposes. [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 543. doi:10.1158/1538-7445.AM2015-543

Abstract 1940: Short (S) isoform of cancer-stem-cell marker, DCLK1, is critically required to maintain proliferative/tumorigenic potential of colon cancer cells: identification of associated molecular pathways

DCLK1 (doublecortin-like-kinase-1) was recently identified as a specific marker for intestinal cancer-stem-cells (CSC) (Sureban et al, 2013; Nakanishi et al, 2013). Importantly, DCLK1 expression is significantly increased during colon-carcinogenesis (Gagliardi et al, 2012). We recently reported that 2 isoforms of DCLK1 (DCLK1-L/DCLK1-S) are transcribed by two different promoters (5′ and intron V), and that the L isoform (∼80KDa) is silenced by DNA methylation in human colon-adenocarcinomas, while the S-isoform (∼47KDa) is up-regulated by many fold in tumors; normal colonic cells mainly express the L-isoform (∼80KDa). Loss of DCLK1 expression in cancer cells/polyps was reported to result in loss of proliferation/polyp formation (Sureban et al 2011; Nakanishi et al, 2013). RNAi methods used so far, target both isoforms of DCLK1. In the current studies we used shRNA to specifically target S-isoform, in order to delineate biological role of cancer-specific S-isoform. Isogenic clones of colon cancer cells, expressing control shRNA (C-clones) or DCLK1-shRNA (KO clones), were generated. Western Blot analysis and RT-PCR analysis confirmed 80-90% knockdown compared to C-clones. Proliferation and Colonogenic/tumorigenic potential of KO clones was completely attenuated compared to C-clones, in vitro and in vivo. Our results confirmed that DCLK1-S is required for maintaining proliferative/tumorigenic potential of colon CSCs. Surprisingly, KO clones did not form spheroids in non-adherent cultures, while the C-clones formed spheroids within 4-6 days of seeding, suggesting DCLK1 may maintain stemness of colon cancer cells. To evaluate molecular pathways mediating effects of DCLK1-S, isogenic C and KO clones were subjected to next generation sequencing (RNAseq). 12 genes were up-regulated and 14 genes were down-regulated in KO vs C clones by >3fold; several others were changed by >1fold. Key genes involved in mitosis, EMT, cell adhesion, caspase-independent apoptosis, metabolism, and toll-like receptor signaling, were significantly down/up-regulated confirming that the S-isoform plays a critical role in CSC biology, by either directly or indirectly regulating several molecular pathways which likely support CSC phenotype. Conclusion. Our results show DCLK1-S is critically required for maintaining invasive/tumorigenic potential of colon cancer cells by directly or indirectly regulating several molecular pathways involved in maintaining non-differentiated/tumorigenic phenotype of CSCs. Thus DCLK1-S may serve as a diagnostic/prognostic marker and provide a useful cancer-specific target for eradicating colon CSCs. This work was supported by NIH Grants to PS (R01CA09795909 and R01CA09795909-S1) Citation Format: Malaney R. O9Connell, Shubhashish Sarkar, Pomila Singh. Short (S) isoform of cancer-stem-cell marker, DCLK1, is critically required to maintain proliferative/tumorigenic potential of colon cancer cells: identification of associated molecular pathways. [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 1940. doi:10.1158/1538-7445.AM2014-1940