Exploiting bioactive natural products of marine origin: Evaluation of the meroterpenoid metachromin V as a novel potential therapeutic drug for colorectal cancer.

Colorectal cancer (CRC) is the second most common cause of cancer death, leading to almost 1 million deaths per year. Despite constant progress in surgical and therapeutic protocols, the 5-year survival rate of advanced CRC patients remains extremely poor. Colorectal Cancer Stem Cells (CRC-CSCs) are endowed with unique stemness-related properties responsible for resistance, relapse and metastasis. The development of novel therapeutics able to tackle CSCs while avoiding undesired toxicity is a major need for cancer treatment. Natural products are a large reservoir of unexplored compounds with possible anticancer bioactivity, sustainability, and safety. The family of meroterpenoids derived from sponges share interesting bioactive properties. Bioassay-guided fractionation of a meroterpenoids extract led to the isolation of three compounds, all cytotoxic against several cancer cell lines: Metachromins U, V and W. In this study, we evaluated the anticancer potential of the most active one, Metachromins V (MV), on patient-derived CRC-CSCs. MV strongly impairs CSCs-viability regardless their mutational background and the cytotoxic effect is maintained on therapy-resistant metastatic CSCs. MV affects cell cycle progression, inducing a block in G2 phase in all the cell lines tested and more pronouncedly in CRC-CSCs. Moreover, MV triggers an important reorganization of the cytoskeleton and a strong reduction of Rho GTPases expression, impairing CRC-CSCs motility and invasion ability. By Proteomic analysis identified a potential molecular target of MV: CCAR1, that regulates apoptosis under chemotherapy treatments and affect ß-catenin pathway. Further studies will be needed to confirm and validate these data in in vivo experimental models.

Proinflammatory and Cancer-Promoting Pathobiont Fusobacterium nucleatum Directly Targets Colorectal Cancer Stem Cells.

Intestinal bacterial communities participate in gut homeostasis and are recognized as crucial in bowel inflammation and colorectal cancer (CRC). Fusobacterium nucleatum (Fn), a pathobiont of the oral microflora, has recently emerged as a CRC-associated microbe linked to disease progression, metastasis, and a poor clinical outcome; however, the primary cellular and/or microenvironmental targets of this agent remain elusive. We report here that Fn directly targets putative colorectal cancer stem cells (CR-CSCs), a tumor cell subset endowed with cancer re-initiating capacity after surgery and chemotherapy. A patient-derived CSC line, highly enriched (70%) for the stem marker CD133, was expanded as tumor spheroids, dissociated, and exposed in vitro to varying amounts (range 100-500 MOI) of Fn. We found that Fn stably adheres to CSCs, likely by multiple interactions involving the tumor-associated Gal-GalNac disaccharide and the Fn-docking protein CEA-family cell adhesion molecule 1 (CEACAM-1), robustly expressed on CSCs. Importantly, Fn elicited innate immune responses in CSCs and triggered a growth factor-like, protein tyrosine phosphorylation cascade largely dependent on CEACAM-1 and culminating in the activation of p42/44 MAP kinase. Thus, the direct stimulation of CSCs by Fn may contribute to microbiota-driven colorectal carcinogenesis and represent a target for innovative therapies.

PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery.

Obesity is a strong risk factor for cancer progression, posing obesity-related cancer as one of the leading causes of death. Nevertheless, the molecular mechanisms that endow cancer cells with metastatic properties in patients affected by obesity remain unexplored.Here, we show that IL-6 and HGF, secreted by tumor neighboring visceral adipose stromal cells (V-ASCs), expand the metastatic colorectal (CR) cancer cell compartment (CD44v6 + ), which in turn secretes neurotrophins such as NGF and NT-3, and recruits adipose stem cells within tumor mass. Visceral adipose-derived factors promote vasculogenesis and the onset of metastatic dissemination by activation of STAT3, which inhibits miR-200a and enhances ZEB2 expression, effectively reprogramming CRC cells into a highly metastatic phenotype. Notably, obesity-associated tumor microenvironment provokes a transition in the transcriptomic expression profile of cells derived from the epithelial consensus molecular subtype (CMS2) CRC patients towards a mesenchymal subtype (CMS4). STAT3 pathway inhibition reduces ZEB2 expression and abrogates the metastatic growth sustained by adipose-released proteins. Together, our data suggest that targeting adipose factors in colorectal cancer patients with obesity may represent a therapeutic strategy for preventing metastatic disease.

Nobiletin and Xanthohumol Sensitize Colorectal Cancer Stem Cells to Standard Chemotherapy.

Colorectal cancer (CRC) mortality is mainly caused by patient refractoriness to common anti-cancer therapies and consequent metastasis formation. Besides, the notorious toxic side effects of chemotherapy are a concurrent obstacle to be tackled. Thus, new treatment approaches are needed to effectively improve patient outcomes. Compelling evidence demonstrated that cancer stem cells (CSCs) are responsible for treatment failure and relapse. New natural treatment approaches showed capabilities to selectively target the CSC subpopulation by rendering them targetable by standard cytotoxic compounds. Herein we show the anti-cancer properties of the polymethoxyflavones and prenylflavonoids extracted from Citrus sinensis and Humulus lupulus, respectively. The natural biofunctional fractions, singularly and in combination, reduced the cell viability of CRC stem cells (CR-CSCs) and synergized with 5-fluorouracil and oxaliplatin (FOX) chemotherapy. These phenomena were accompanied by a reduced S and G2/M phase of the cell cycle and upregulation of cell death-related genes. Notably, both phytoextracts in combination with FOX thwarted stemness features in CR-CSCs as demonstrated by the impaired clonogenic potential and decreased Wnt pathway activation. Extracts lowered the expression of CD44v6 and affected the expansion of metastatic CR-CSCs in patients refractory to chemotherapy. Together, this study highlights the importance of polymethoxyflavones and prenylflavonoids as natural remedies to aid oncological therapies.

CHK1 inhibitor sensitizes resistant colorectal cancer stem cells to nortopsentin.

Limited therapeutic options are available for advanced colorectal cancer (CRC). Herein, we report that exposure to a neo-synthetic bis(indolyl)thiazole alkaloid analog, nortopsentin 234 (NORA234), leads to an initial reduction of proliferative and clonogenic potential of CRC sphere cells (CR-CSphCs), followed by an adaptive response selecting the CR-CSphC-resistant compartment. Cells spared by the treatment with NORA234 express high levels of CD44v6, associated with a constitutive activation of Wnt pathway. In CR-CSphC-based organoids, NORA234 causes a genotoxic stress paralleled by G2-M cell cycle arrest and activation of CHK1, driving the DNA damage repair of CR-CSphCs, regardless of the mutational background, microsatellite stability, and consensus molecular subtype. Synergistic combination of NORA234 and CHK1 (rabusertib) targeting is synthetic lethal inducing death of both CD44v6-negative and CD44v6-positive CRC stem cell fractions, aside from Wnt pathway activity. These data could provide a rational basis to develop an effective strategy for the treatment of patients with CRC.

Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells.

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients' prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.

miR-1285-3p Controls Colorectal Cancer Proliferation and Escape from Apoptosis through DAPK2.

MicroRNAs are tiny but powerful regulators of gene expression at the post-transcriptional level. Aberrant expression of oncogenic and tumor-suppressor microRNAs has been recognized as a common feature of human cancers. Colorectal cancer represents a major clinical challenge in the developed world and the design of innovative therapeutic approaches relies on the identification of novel biological targets. Here, we perform a functional screening in colorectal cancer cells using a library of locked nucleic acid (LNA)-modified anti-miRs in order to unveil putative oncogenic microRNAs whose inhibition yields a cytotoxic effect. We identify miR-1285-3p and further explore the effect of its targeting in both commercial cell lines and primary colorectal cancer stem cells, finding induction of cell cycle arrest and apoptosis. We show that DAPK2, a known tumor-suppressor, is a novel miR-1285 target and mediates both the anti-proliferative and the pro-apoptotic effects of miR-1285 depletion. Altogether, our findings uncover a novel oncogenic microRNA in colorectal cancer and lay the foundation for further studies aiming at the development of possible therapeutic strategies based on miR-1285 targeting.

Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance.

In the last decades, the role of the microenvironment in tumor progression and therapeutic outcome has gained increasing attention. Cancer-associated fibroblasts (CAFs) have emerged as key players among stromal cells, owing to their abundance in most solid tumors and their diverse tumor-restraining/promoting roles. The interplay between tumor cells and neighboring CAFs takes place by both paracrine signals (cytokines, exosomes and metabolites) or by the multifaceted functions of the surrounding extracellular matrix. Here, we dissect the most recent identified mechanisms underlying CAF-mediated control of tumor progression and therapy resistance, which include induction of the epithelial-to-mesenchymal transition (EMT), activation of survival pathways or stemness-related programs and metabolic reprogramming in tumor cells. Importantly, the recently unveiled heterogeneity in CAFs claims tailored therapeutic efforts aimed at eradicating the specific subset facilitating tumor progression, therapy resistance and relapse. However, despite the large amount of pre-clinical data, much effort is still needed to translate CAF-directed anti-cancer strategies from the bench to the clinic.