Loss of Myosin Vb in colorectal cancer is a strong prognostic factor for disease recurrence

Background: Selecting the most beneficial treatment regimens for colorectal cancer (CRC) patients remains challenging due to a lack of prognostic markers. Members of the Myosin family, proteins recognized to play a major role in trafficking and polarization of cells, have recently been reported to be closely associated with several types of cancer and might thus serve as potential prognostic markers in the context of CRC. Methods: We used a previously established meta-analysis of publicly available gene expression data to analyse the expression of different members of the Myosin V family, namely MYO5A, 5B, and 5C, in CRC. Using laser-microdissected material as well as tissue microarrays from paired human CRC samples, we validated both RNA and protein expression of MYO5B and its known adapter proteins (RAB8A and RAB25) in an independent patient cohort. Finally, we assessed the prognostic value of both MYO5B and its adapter-coupled combinatorial gene expression signatures. Results: The meta-analysis as well as an independent patient cohort study revealed a methylation-independent loss of MYO5B expression in CRC that matched disease progression. Although MYO5B mutations were identified in a small number of patients, these cannot be solely responsible for the common down-regulation observed in CRC patients. Significantly, CRC patients with low MYO5B expression displayed shorter overall, disease- and metastasis-free survival, a trend that was further reinforced when RAB8A expression was also taken into account. Conclusions: Our data identifies MYO5B as a powerful prognostic biomarker in CRC, especially in early stages (stages I and II), which might help stratifying patients with stage II for adjuvant chemotherapy

Hypoxia-induced Autophagy Drives Colorectal Cancer Initiation and Progression by Activating the PRKC/PKC-EZR (Ezrin) Pathway

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enriched patient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

SC are more resistant to 5-FU treatment.

<p>(<i>a</i>) Experimental layout of the sphere formation and chemosensitivity assay. (<i>b</i>) Relative sphere size of SC and their differentiated counterparts exposed to different concentrations of 5-FU over 5 days. Representative figure of 2 independent experiments. Statistics are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146052#pone.0146052.s012" target="_blank">S5A–S5D Table</a>. (<i>c</i>) Relative survival of SC and their differentiated counterparts after exposure to 50 μM 5-FU for 5 days. Representative figure of 2 independent experiments is shown. Data are presented as mean ± SD (n = 8), *P<0.05, **P<0.01, ***P<0.001, ns = not significant. (<i>d-e</i>) Clonogenic and self-renewal capacity of SC and adherent differentiated counterparts were assessed by performing colony- or sphere-formation assays respectively. 5FU (5μM) was used to assess chemoresistance in both culture types. For T6, 2 independent experiments were performed whereas one single experiment was performed for HT29.</p

SC are highly tumorigenic but do not show differences in tumor incidence compared to adherent differentiated counterparts.

<p>(<i>a</i>) Primary SC induce tumors in mice. (<i>b</i>) Tumor weight of the generated tumor xenografts from primary SC. (<i>c</i>) Xenograft derived from primary SC recapitulate the phenotype of the primary tumor after serial transplantation in mice. Magnification 10x. (<i>d</i>) Tumor growth in mice after injection of SC and their differentiated counterpart. Tumor incidence is indicated within the graph. Data are presented as mean ± SEM, *P<0.05.</p

Gene signature derived from SC predicts poor outcome in CRC patients.

<p>(<i>a-b</i>) An overlap of 8 commonly up-regulated genes for patient and cell line-derived SC (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146052#pone.0146052.s013" target="_blank">S6 Table</a>) predicts for overall survival in CRC. (<i>b</i>) The combined expression of two genes, <i>CDA</i> & <i>GSTA4</i>, is linked to an increased risk of disease relapse. The number of patients in each group is mentioned within the brackets; significant p-value is indicated.</p

Hypoxia-responsive miR-210 promotes self-renewal capacity of colon tumor-initiating cells by repressing ISCU and by inducing lactate production

Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor-initiating cells (TICs). To better understand the mechanism of hypoxia-induced TIC activation, we set out to study the role of hypoxia-responsive miRNAs in recently established colon cancer patientderived TICs. We were able to show that low oxygen concentrations consistently lead to the upregulation of miR-210 in different primary TIC-enriched cultures. Both stable overexpression of miR-210 and knockdown of its target gene ISCU resulted in enhanced TIC self-renewal. We could validate the tumorigenic properties of miR- 210 in in vivo experiments by showing that ectopic expression of miR-210 results in increased tumor incidence. Furthermore, enhanced miR-210 expression correlated with reduced TCA cycle activity and increased lactate levels. Importantly, by blocking lactate production via inhibition of LDHA, we could reverse the promoting effect of miR-210 on self-renewal capacity, thereby emphasizing the regulatory impact of the glycolytic phenotype on colon TIC properties. Finally, by assessing expression levels in patient tissue, we could demonstrate the clinical relevance of the miR-210/ISCU signaling axis for colorectal carcinoma. Taken together, our study highlights the importance of hypoxia-induced miR-210 in the regulation of colon cancer initiation

Self-renewal capacity is maintained over several passages.

<p>(<i>a</i>) Sphere formation rates were determined over several generations (gen.) by seeding single cells of early passage SC grown from primary tumor tissue and CRC cell lines. Data are presented as mean ± SD. (<i>b</i>) Self-renewal capacity of early (passage 5) and late (passage 15–25 depending on the tumor) SC from primary tumor tissue. Data are presented as mean with 95% confidence interval.</p

The microRNA-371~373 cluster represses colon cancer initiation and metastatic colonization by inhibiting the TGFBR2/ID1 signaling axis.

The vast majority of colorectal cancer (CRC)-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different CRC cell lines and recently established primary cultures enriched in colon cancer stem cells (CSCs) - also known as tumor-initiating cells (TICs) - to identify genes and microRNAs (miRNAs) with regulatory functions in CRC progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, transforming growth factor beta (TGF-beta) signaling activity, and reduced expression of the miR-371~373 cluster compared to non-metastatic cultures. TGF-beta receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371~373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371~373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor-initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371~373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371~373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371~373 and ID1. Altogether, our results establish the miR-371~373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization