Mutational signatures in colon cancer.

ObjectiveRecently, many tumor sequencing studies have inferred and reported on mutational signatures, short nucleotide patterns at which particular somatic base substitutions appear more often. A number of signatures reflect biological processes in the patient and factors associated with cancer risk. Our goal is to infer mutational signatures appearing in colon cancer, a cancer for which environmental risk factors vary by cancer subtype, and compare the signatures to those in adult stem cells from normal colon. We also compare the mutational signatures to others in the literature.ResultsWe apply a probabilistic mutation signature model to somatic mutations previously reported for six adult normal colon stem cells and 431 colon adenocarcinomas. We infer six mutational signatures in colon cancer, four being specific to tumors with hypermutation. Just two signatures explained the majority of mutations in the small number of normal aging colon samples. All six signatures are independently identified in a series of 295 Chinese colorectal cancers

Synthesis of 4-thio-5-(2′′-thienyl)uridine and cytotoxicity activity against colon cancer cells <i>in vitro</i>

A novel anti-tumor agent 4-thio-5-(2′′-thienyl)uridine (6) was synthesized and the in vitro cytotoxicity activity against mice colon cancer cells (MC-38) and human colon cancer cells (HT-29) was evaluated by MTT assay. The results showed that the novel compound had antiproliferative activity toward MC-38 and HT-29 cells in a dose-dependent manner. The cell cycle analysis by flow cytometry indicated that compound 6 exerted in tumor cell proliferation inhibition by arresting HT-29 cells in the G2/M phase. In addition, cell death detected by propidium iodide staining showed that compound 6 efficiently induced cell apoptosis in a concentration-dependent manner. Moreover, the sensitivity of human fibroblast cells to compound 6 was far lower than that of tumor cells, suggesting the specific anti-tumor effect of 4-thio-5-(2′′-thienyl)uridine. Taken together, novel compound 6 effectively inhibits colon cancer cell proliferation, and hence would have potential value in clinical application as an antitumor agent

Anticancer Activities of Meroterpenoids Isolated from the Brown Alga Cystoseira usneoides against the Human Colon Cancer Cells HT-29

Colorectal cancer (CRC) is one of the most common types of cancers and a leading cause of cancer death worldwide. The current treatment for CRC mainly involves surgery, radiotherapy, and chemotherapy. However, due to the side effects and the emergence of drug resistance, the search for new anticancer agents, pharmacologically safe and effective, is needed. In the present study, we have investigated the anticancer effects of eight algal meroterpenoids (AMTs, 1-8) isolated from the brown seaweed Cystoseira usneoides and their underlying mechanisms of action using HT-29, a highly metastatic human colon cancer cell line. All the tested meroterpenoids inhibited the growth of HT-29 malignant cells and were less toxic towards non-cancer colon cells, with the AMTs 1 and 5 exhibiting selectivity indexes of 5.26 and 5.23, respectively. Treatment of HT-29 cells with the AMTs 1, 2, 3, 4, 5, and 7 induced cell cycle arrest in G2/M phase and, in some instances, apoptosis (compounds 2, 3, and 5). Compounds 1-8 also exhibited significant inhibitory effects on the migration and/or invasion of colon cancer cells. Mechanistic analysis demonstrated that the AMTs 1, 2, 5, 6, 7, and 8 reduced phosphorylation levels of extracellular signal-regulated kinase (ERK) and the AMTs 2, 3, 4, 5, 7, and 8 decreased phosphorylation of c-JUN N-terminal kinase (JNK). Moreover, the AMTs 1, 2, 3, 4, 7, and 8 inhibited phosphorylation levels of protein kinase B (AKT) in colon carcinoma cells. These results provide new insights into the mechanisms and functions of the meroterpenoids of C. usneoides, which exhibit an anticancer effect on HT-29 colon cancer cells by inducing cell cycle arrest and apoptosis via the downregulation of ERK/JNK/AKT signaling pathways

Prospectively Isolated Cancer-Associated CD10+ Fibroblasts Have Stronger Interactions with CD133+ Colon Cancer Cells than with CD133− Cancer Cells

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133+ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133+ and CD133− colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10+ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133+ and CD133− subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133+ cells showed significantly greater tumor growth than CD133− cells (P = 0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133+ cells exhibited significantly more invasive behaviors than CD133− cells (P<0.001), especially in cocultures with CD10+ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10+ fibroblasts enhanced the tumor growth of CD133+ cells significantly more than CD10− fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133+ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10+ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies

Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models.

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24-31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery

Role of novel tumor suppressors in colon cancer : Mechanisms and therapeutic opportunities

Colon cancer is the third most common cancer in the world and the fourth most common cause of cancer related deaths. Inflammation is one of the risk factors for development of colon cancer. Interestingly immune cells as mast cells and inflammatory mediators as LTC4 play an important role in colon cancer. Genetic predisposition, which might lead to either activation of oncogenes or inhibition of tumor suppressor genes, are risk factors of colon cancer development.The aim of my thesis was to evaluate the clinical significance of the tumor suppressors 15-PGDH and WNT5A in colon cancer patients. I investigated the underlying mechanisms/signaling triggered by these tumor suppressors in colon cancer cells and whether the re-expression of these tumor suppressors could be an attractive therapeutic strategy for treatment of colon cancer patients.I found that presence of mast cells in colon cancer tissue was associated with better prognosis of colon cancer patients, and the presence of mast cells in polyps/tumors in a colitis-associated colon cancer mouse model was also beneficial. I found that the tumor suppressor gene 15-PGDH is down-regulated in colon cancer patients as well as in colon cancer cell lines. This down- regulation is often seen in parallel with down-regulated WNT5A, the non-canonical Wnt/β-catenin signaling ligand. I found that down-regulation of both these proteins is associated with poor prognosis for colon cancer patients. My results show that treatment of colon cancer cells with Foxy-5, a WNT5A mimicking peptide, leads to up-regulation of 15-PGDH through JNK/AP-1 pathway. In addition, I also found that the pro-inflammatory mediator LTC4 via CysLTR2 can induce the expression of 15-PGDH through the JNK pathway, which indicate that LTC4 via CysLTR2 has an anti-tumor effect.In conclusion, these findings provide important information for better understanding of the tumor microenvironment and the tumor suppressor genes in colon cancer and might help to identify new therapeutic targets for colon cancer patients

Adipocytes Activate Mitochondrial Fatty Acid Oxidation and Autophagy to Promote Tumor Growth in Colon Cancer

Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells

The glycosphingolipid globotriaosylceramide in the metastatic transformation of colon cancer

The most devastating aspect of cancer is the emergence of metastases. Thus, identification of potentially metastatic cells among a tumor cell population and the underlying molecular changes that switch cells to a metastatic state are among the most important issues in cancer biology. Here we show that, although normal human colonic epithelial cells lack the glycosphingolipid globotriaosylceramide (Gb3), this molecule is highly expressed in metastatic colon cancer. In addition, a subpopulation of cells that are greatly enriched in Gb3 and have an invasive phenotype was identified in human colon cancer cell lines. In epithelial cells in culture, Gb3 was necessary and sufficient for cell invasiveness. Transfection of Gb3 synthase, resulting in Gb3 expression in noncancerous polarized epithelial cells lacking endogenous Gb3, induced cell invasiveness. Furthermore, Gb3 knockdown by small inhibitory RNA in colon cancer epithelial cells inhibited cell invasiveness. Gb3 is the plasma membrane receptor for Shiga toxin 1. The noncatalytic B subunit of Shiga toxin 1 causes apoptosis of human colon cancer cells expressing Gb3. Injections of the B subunit of Shiga toxin 1 into HT29 human colon cancer cells engrafted into the flanks of nude mice inhibited tumor growth. These data demonstrate the appearance of a subpopulation of Gb3 containing epithelial cells in the metastatic stage of human colon cancer and suggest their possible role in colon cancer invasiveness

HMGA1 Induces Intestinal Polyposis in Transgenic Mice and Drives Tumor Progression and Stem Cell Properties in Colon Cancer Cells

Although metastatic colon cancer is a leading cause of cancer death worldwide, the molecular mechanisms that enable colon cancer cells to metastasize remain unclear. Emerging evidence suggests that metastatic cells develop by usurping transcriptional networks from embryonic stem (ES) cells to facilitate an epithelial-mesenchymal transition (EMT), invasion, and metastatic progression. Previous studies identified HMGA1 as a key transcription factor enriched in ES cells, colon cancer, and other aggressive tumors, although its role in these settings is poorly understood.To determine how HMGA1 functions in metastatic colon cancer, we manipulated HMGA1 expression in transgenic mice and colon cancer cells. We discovered that HMGA1 drives proliferative changes, aberrant crypt formation, and intestinal polyposis in transgenic mice. In colon cancer cell lines from poorly differentiated, metastatic tumors, knock-down of HMGA1 blocks anchorage-independent cell growth, migration, invasion, xenograft tumorigenesis and three-dimensional colonosphere formation. Inhibiting HMGA1 expression blocks tumorigenesis at limiting dilutions, consistent with depletion of tumor-initiator cells in the knock-down cells. Knock-down of HMGA1 also inhibits metastatic progression to the liver in vivo. In metastatic colon cancer cells, HMGA1 induces expression of Twist1, a gene involved in embryogenesis, EMT, and tumor progression, while HMGA1 represses E-cadherin, a gene that is down-regulated during EMT and metastatic progression. In addition, HMGA1 is among the most enriched genes in colon cancer compared to normal mucosa.Our findings demonstrate for the first time that HMGA1 drives proliferative changes and polyp formation in the intestines of transgenic mice and induces metastatic progression and stem-like properties in colon cancer cells. These findings indicate that HMGA1 is a key regulator, both in metastatic progression and in the maintenance of a stem-like state. Our results also suggest that HMGA1 or downstream pathways could be rational therapeutic targets in metastatic, poorly differentiated colon cancer

Inhibition of ubiquitin specific peptidase 8 is effective against 5-fluorouracil resistance in colon cancer via suppressing EGFR and EGFR-mediated signaling pathways

Background. The identification of a sensitizing strategy to overcome 5-fluorouracil (5-FU) therapeutic resistance is needed in colon cancer. Recent studies highlight the oncogenic role of ubiquitin specific peptidase 8 (USP8) in many cancers. In line with these efforts, this work investigated the therapeutic potential of targeting USP8 in colon cancer. Methods. Immunohistochemistry was performed to determine USP8 expression level in colon cancer tissues and their adjacent normal tissues. Gain-of-function analysis via plasmid overexpression and loss-of-function analysis via siRNA knockdown were applied on cellular assays. The combinatory effects of USP8 inhibitor and cisplatin were determined using a colon xenograft mouse model. Immunoblotting was performed to investigate the molecular mechanism of USP8 inhibition in colon cancer cells. Results. Compared to normal counterparts, we showed that USP8 protein level was significantly higher in colon cancer tissues and cells. In addition, USP8 expression was not affected by prolonged exposure of colon cancer cells to 5-FU. USP8 was important for colon cancer cell growth and survival but not migration as assessed by loss-of-function and gain-of-function approaches. Pharmacological inhibition of USP8 using USP8 inhibitor is active against both sensitive and 5-FUresistant colon cancer cells. Of note, USP8 inhibitor significantly inhibited colon cancer formation and growth, and augmented in vivo efficacy of 5-FU without causing toxicity in mice. Mechanistic studies showed that USP8 inhibitor acted on colon cancer cells through suppressing EGFR and EGFR-mediated signalling pathways. Conclusions. Our work is the first to reveal the essential role of USP8 in colon cancer via EGFR oncogenic signalling pathways. Our findings provide a proof-of-concept that USP8 inhibitors are promising candidates to overcome 5-FU resistance in colon cance