Hypoxic Colorectal Cancer Research Articles

Chloroquine sensitises hypoxic colorectal cancer cells to ROS-mediated cell death via structural disruption of pyruvate dehydrogenase kinase 1.

Chloroquine (CQ), an autophagy antagonist, has been recently explored as a repurposable medicine for cancer; however the exact mechanism of its action is still not known. The present study investigated the effect of CQ on colorectal cancer cells to elucidate the underlying molecular mechanisms. We report for the first time that CQ suppresses hypoxia-induced growth and survival of HCT-116 cells by reducing glycolytic capacity and NAD+ production through inhibition of PDK1. Furthermore, in silico and in vitro studies revealed that CQ induces structural alteration in the PDK1 protein, leading to its destabilization and promotes its enhanced degradation by proteases. This degradation is in turn inhibited by the MG-132 protease inhibitor. Moreover, CQ-induced suppression of PDK1 results in mitochondrial damage through excessive production of ROS, as reflected by the reduction in mitochondrial membrane potential, which in turn triggers apoptosis through PARP cleavage and Caspase activation. These findings advocate CQ as a promising repurposable chemotherapeutic for colorectal cancer and a novel inhibitor of PDK1.

Fructose shields human colorectal cancer cells from hypoxia-induced necroptosis

Recent studies have shown that high dietary fructose intake enhances intestinal tumor growth in mice. Our previous work indicated that glucose enables hypoxic colorectal cancer (CRC) cells to resist receptor-interacting protein (RIP)-dependent necroptosis. Despite having the same chemical formula, glucose and fructose are absorbed through different transporters yet both can enter the glycolytic metabolic pathway. The excessive intake of dietary fructose, leading to its overflow into the colon, allows colonic cells to absorb fructose apically. This study explores the mechanisms behind apical fructose-mediated death resistance in CRC cells under hypoxic stress. Utilizing three CRC cell lines (Caco-2, HT29, and T84) under normoxic and hypoxic conditions with varying fructose concentrations, we assessed lactate dehydrogenase (LDH) activity, RIP1/3 complex formation (a necroptosis marker), and cell integrity. We investigated the role of fructose in glycolytic-mediated death resistance using glycolytic inhibitors iodoacetate (IA, a glycolytic inhibitor to glyceraldehyde 3-phosphate dehydrogenase), and UK5099 (UK, an inhibitor to mitochondrial pyruvate carrier). Our findings reveal that apical fructose prevents the hypoxia-induced RIP-dependent necroptosis in Caco-2 and HT29 cells. Fructose exposure under hypoxia also preserved epithelial integrity. IA, but not UK, blocked fructose-mediated glycolytic metabolite production and necrosis, indicating that anaerobic glycolytic metabolites facilitate death resistance. Notably, fructose treatment upregulated pyruvate kinase (PK)-M1 mRNA in hypoxic Caco-2 and HT29 cells, while PKM2 upregulation was exclusive to HT29 cells. In conclusion, apical fructose utilization through glycolysis effectively inhibits hypoxia-induced RIP-dependent necroptosis in CRC cells, shedding light on potential metabolic adaptation mechanisms in the tumor microenvironment and suggesting novel targets for therapeutic intervention.

Resveratrol can Reduce the Aggressiveness of Hypoxic Colon Cancer Cells

Colorectal cancer is a significant global health problem characterized by the development of metastasis due to fast cell growth, tolerance to low oxygen levels, and the formation of new blood vessels. Notable advancements have been seen in the management of these instances; however, uncertainties persist about drug resistance and its accompanying adverse effects. Resveratrol, a natural polyphenol derived from several plants, has diverse pharmacological characteristics. The anticancer impact of this has piqued the curiosity of several researchers. The objective of our research was to investigate the impact of resveratrol on the proliferation, migration, and expression of angiogenic factors in hypoxic colorectal cancer cells by the use of resveratrol. Hypoxia was chemically induced using cobalt chloride. Serially diluted concentrations of resveratrol (200, 100, 50, 25, 12.5, and 6.25 \(\mu\)g/ml) were employed to assess the cytotoxic effect through the MTT assay. Smaller concentrations (below IC50) were utilized to investigate the impact of resveratrol on the migration of SW480 cells using the wound healing assay (50 \(\mu\)g/ml). The impact of resveratrol on the expression of vascular endothelial growth factor (VEGF) was assessed using the enzyme-linked immunosorbent assay (ELISA). The findings shown that resveratrol has the ability to effectively decrease cell proliferation in a dose-dependent way, inhibit cell migration and angiogenesis, via suppression of VEGF and HIF-1\(\alpha\). The significance of this etude lies in the enduring inability to effectively manage metastatic colorectal cancer. Suggesting that resveratrol might function as an adjunctive therapy and a useful supplement for those suffering from very metastatic colorectal cancer. The specific method by which resveratrol works is yet unknown, hence more study is needed to conduct experiments in living organisms and clinical trials.

Anti-Cancer Study of Carvacrol in Hypoxic-Induced Colorectal Cancer Cell

Introduction: Colorectal cancer presents a complex global health challenge, characterized by complications arising from metastasis development due to rapid proliferation, adaptation to hypoxia, and angiogenesis. Carvacrol, a natural monoterpenoid phenol derived from various plants, has interest for its diverse pharmacological properties, particularly its antitumor effects. Methods: We aimed to assess the inhibitory effect of carvacrol on cell migration and proliferation in the hypoxic colorectal cancer cell line (SW480). Chemical induction of hypoxia using cobalt chloride and serially diluted concentrations of carvacrol (400, 200, 100, 50, 25, and 12.5 µg/ml) were employed to evaluate the cytotoxic effect via the MTT assay. Smaller concentrations (low IC50) were used to examine the impact of carvacrol on SW480 cell migration through a cell migration assay (50, 25, 12.5, and 6.25 µg/ml). Results: The results demonstrated a significant, dose-dependent reduction in both cell proliferation and migration with carvacrol doses. Conclusion: The findings suggest that carvacrol could be useful as an adjuvant therapy and a promising addition for patients with highly metastatic colorectal cancer.

Dual-driven nanomotors enable tumor penetration and hypoxia alleviation for calcium overload-photo-immunotherapy against colorectal cancer

The treatment efficacies of conventional medications against colorectal cancer (CRC) are restricted by a low penetrative, hypoxic, and immunosuppressive tumor microenvironment. To address these restrictions, we developed an innovative antitumor platform that employs calcium overload-phototherapy using mitochondrial N770-conjugated mesoporous silica nanoparticles loaded with CaO2 (CaO2–N770@MSNs). A loading level of 14.0 wt% for CaO2–N770@MSNs was measured, constituting an adequate therapeutic dosage. With the combination of oxygen generated from CaO2 and hyperthermia under near-infrared irradiation, CaO2–N770@MSNs penetrated through the dense mucus, accumulated in the colorectal tumor tissues, and inhibited tumor cell growth through endoplasmic reticulum stress and mitochondrial damage. The combination of calcium overload and phototherapy revealed high therapeutic efficacy against orthotopic colorectal tumors, alleviated the immunosuppressive microenvironment, elevated the abundance of beneficial microorganisms (e.g., Lactobacillaceae and Lachnospiraceae), and decreased harmful microorganisms (e.g., Bacteroidaceae and Muribaculaceae). Moreover, together with immune checkpoint blocker (αPD-L1), these nanoparticles showed an ability to eradicate both orthotopic and distant tumors, while potentiating systemic antitumor immunity. This treatment platform (CaO2–N770@MSNs plus αPD-L1) open a new horizon of synergistic treatment against hypoxic CRC with high killing power and safety.

Hypoxia induces downregulation of the tumor-suppressive sST2 in colorectal cancer cells via the HIF–nuclear IL-33–GATA3 pathway

As a decoy receptor, soluble ST2 (sST2) interferes with the function of the inflammatory cytokine interleukin (IL)-33. Decreased sST2 expression in colorectal cancer (CRC) cells promotes tumor growth via IL-33-mediated bioprocesses in the tumor microenvironment. In this study, we discovered that hypoxia reduced sST2 expression in CRC cells and explored the associated molecular mechanisms, including the expression of key regulators of ST2 gene transcription in hypoxic CRC cells. In addition, the effect of the recovery of sST2 expression in hypoxic tumor regions on malignant progression was investigated using mouse CRC cells engineered to express sST2 in response to hypoxia. Our results indicated that hypoxia-dependent increases in nuclear IL-33 interfered with the transactivation activity of GATA3 for ST2 gene transcription. Most importantly, hypoxia-responsive sST2 restoration in hypoxic tumor regions corrected the inflammatory microenvironment and suppressed tumor growth and lung metastasis. These results indicate that strategies targeting sST2 in hypoxic tumor regions could be effective for treating malignant CRC.

Repurposing Sulfasalazine as a Radiosensitizer in Hypoxic Human Colorectal Cancer.

xCT overexpression in cancer cells has been linked to tumor growth, metastasis and treatment resistance. Sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid sarthritis, and inflammatory bowel diseases, has anticancer properties via inhibition of xCT, leading to the disruption of redox homeostasis. Since reactive oxygen species (ROS) are pivotal for the efficacy of radiotherapy (RT), elevated levels of ROS are associated with improved RT outcomes. In this study, the influence of SSZ treatment on the radiosensitivity of human colorectal cancer (CRC) cells was investigated. Our principal finding in human HCT116 and DLD-1 cells was that SSZ enhances the radiosensitivity of hypoxic CRC cells but does not alter the intrinsic radiosensitivity. The radiosensitizing effect was attributed to the depletion of glutathione and thioredoxin reductase levels. In turn, the reduction leads to excessive levels of ROS, increased DNA damage, and ferroptosis induction. Confirmation of these findings was performed in 3D models and in DLD-1 xenografts. Taken together, this study is a stepping stone for applying SSZ as a radiosensitizer in the clinic and confirms that xCT in cancer cells is a valid radiobiological target.

Upregulation of HMGB1 in tumor-associated macrophages induced by tumor cell-derived lactate further promotes colorectal cancer progression

BackgroundLactate accumulation leads to an acidic tumor microenvironment (TME), in turn promoting colorectal cancer (CRC) progression. Tumor-associated macrophages (TAMs) are the predominant cells in TME. This study aimed to reveal the regulation mechanism of CRC cell-derived lactate on TAMs and explore the mechanism underlying lactate accumulation-induced aggravation in CRC.MethodsCell growth and metastasis were evaluated by colony formation, Transwell, and wound healing assays. Western blot and RT-qPCR were applied to determine the protein and mRNA expression. Flow cytometry was used to analyze the polarization state and apoptotic rate of macrophages induced in THP-1 cells. The lactate in the cell supernatant was quantified using an ELISA kit. Immunofluorescence was performed to visualize the location of High Mobility Group Box 1 (HMGB1). H&E and Ki67 staining assays were used to assess tumorigenesis in nude mice bearing ectopic tumors.ResultsCell growth and metastasis were promoted in the hypoxic CRC cells. The hypoxic cell supernatant stimulated the M2-type polarization of macrophages. The lactate level increased in hypoxic cancer cells. However, the inhibition of lactate using 3-hydroxy-butyrate (3-OBA) reversed the effects of hypoxia. Also, macrophages showed no promoting effect on cancer cell growth and migration in the presence of 3-OBA. HMGB1 was secreted into the extracellular space of lactate-induced macrophages, further enhancing the malignant behaviors of cancer cells. ERK, EMT, and Wnt signaling pathways were activated in cancer cells due to HMGB1 upregulation.ConclusionsThe lactate metabolized by cancer cells stimulated M2 polarization and HMGB1 secretion by macrophages, aggravating the carcinogenic behaviors of cancer cells.

α‑hederin overcomes hypoxia‑mediated drug resistance in colorectal cancer by inhibiting the AKT/Bcl2 pathway.

Currently, chemoresistance is a major challenge that directly affects the prognosis of patients with colorectal cancer (CRC). In addition, hypoxia is associated with poor prognosis and therapeutic resistance in patients with cancer. Accumulating evidence has shown that α‑hederin has significant antitumour effects and that α‑hederin can inhibit hypoxia‑mediated drug resistance in CRC; however, the underlying mechanism remains unclear. In the present study, viability and proliferation assays were used to evaluate the effect of α‑hederin on the drug resistance of CRC cells under hypoxia. Sequencing analysis and apoptosis assays were used to determine the effect of α‑hederin on apoptosis under hypoxia. Western blot analysis and reverse transcription‑quantitative PCR were used to measure apoptosis‑related protein and mRNA expression levels. Furthermore, different mouse models were established to study the effect of α‑hederin on hypoxia‑mediated CRC drug resistance invivo. In the present study, the high expression of Bcl2 in hypoxic CRC cells was revealed to be a key factor in their drug resistance, whereas α‑hederin inhibited the expression of Bcl2 by reducing AKT phosphorylation invitro and invivo, and promoted the apoptosis of CRC cells under hypoxia. By contrast, overexpression of AKT reversed the effect of α‑hederin on CRC cell apoptosis under hypoxia. Taken together, these results suggested that α‑hederin may overcome hypoxia‑mediated drug resistance in CRC by inhibiting the AKT/Bcl2 pathway. In the future, α‑hederin may be used as a novel adjuvant for reversing drug resistance in CRC.

Inhibition of Phosphoglycerate Dehydrogenase Radiosensitizes Human Colorectal Cancer Cells under Hypoxic Conditions.

Simple SummaryColorectal cancer is the third most prevalent cancer worldwide. Treatment options for these patients consist of surgery combined with chemotherapy and/or radiotherapy. However, a subset of tumors will not respond to therapy or acquire resistance during the course of the treatment, leading to patient relapse. The interplay between reprogramming cancer metabolism and radiotherapy has become an appealing strategy to improve a patient’s outcome. Due to the overexpression of certain enzymes in a variety of cancer types, including colorectal cancer, the serine synthesis pathway has recently become an attractive metabolic target. We demonstrated that by inhibiting the first enzyme of this pathway, namely phosphoglycerate dehydrogenase (PHGDH), tumor cells that are deprived of oxygen (as is generally the case in solid tumors) respond better to radiation, leading to increased tumor cell killing in an experimental model of human colorectal cancer. Augmented de novo serine synthesis activity is increasingly apparent in distinct types of cancers and has mainly sparked interest by investigation of phosphoglycerate dehydrogenase (PHGDH). Overexpression of PHGDH has been associated with higher tumor grade, shorter relapse time and decreased overall survival. It is well known that therapeutic outcomes in cancer patients can be improved by reprogramming metabolic pathways in combination with standard treatment options, for example, radiotherapy. In this study, possible metabolic changes related to radioresponse were explored upon PHGDH inhibition. Additionally, we evaluated whether PHGDH inhibition could improve radioresponse in human colorectal cancer cell lines in both aerobic and radiobiological relevant hypoxic conditions. Dysregulation of reactive oxygen species (ROS) homeostasis and dysfunction in mitochondrial energy metabolism and oxygen consumption rate were indicative of potential radiomodulatory effects. We demonstrated that PHGDH inhibition radiosensitized hypoxic human colorectal cancer cells while leaving intrinsic radiosensitivity unaffected. In a xenograft model, the first hints of additive effects between PHGDH inhibition and radiotherapy were demonstrated. In conclusion, this study is the first to show that modulation of de novo serine biosynthesis enhances radioresponse in hypoxic colorectal cancer cells, mainly mediated by increased levels of intracellular ROS.

Forkhead box A2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced epithelial-mesenchymal transition by targeting c14orf28 in colorectal cancer

Background and Study AimsThis study aimed to investigate the effect of Forkhead Box A2 (FOXA2) on migration, invasion, and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells in hypoxia and explore its related molecular mechanisms. Patients and MethodsA cellular hypoxia model was established, and the FOXA2 overexpression vector was transfected into SW480 and HCT116 cells. Cell apoptosis, migration, and invasion were examined by flow cytometry, scratch test, and transwell-invasion assay. Next, the hsa-let-7 g gene expression was detected by quantitative reverse transcription-polymerase chain reaction. Relative protein levels of HIF-1, FOXA2, c14orf28, E-cadherin, N-cadherin, and Vimentin were detected by western blot. ResultsHypoxia-exposed CRC cells showed a significantly increased cell apoptosis rate, as well as enhanced cell invasion and migration abilities compared with the cells in normoxia. FOXA2 overexpression induced apoptosis and inhibited hypoxia-exposed CRC cell migration and invasion. Additionally, FOXA2 overexpression led to the significantly increased hsa-let-7 g and E-cadherin expression, as well as the decreased c14orf28, N-cadherin, and Vimentin expression in hypoxic CRC cells. ConclusionsThis study demonstrated that FOXA2 could affect the apoptosis, migration, invasion, and EMT of CRC cells under hypoxia conditions. FOXA2 transcriptionally activates hsa-let-7 g to inhibit hypoxia-induced EMT by targeting c14orf28.

MO-0141 Sulfasalazine radiosensitizes hypoxic colorectal cancer cells through the inhibition of xCT.

MO-0141 Sulfasalazine radiosensitizes hypoxic colorectal cancer cells through the inhibition of xCT.

Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase.

The major adaptive response to hypoxia involves hypoxia-inducible factor HIF-1α which is regulated by von Hippel Lindau (VHL) E3 ligase. We previously observed a stabilization of HIF-1α by cyclin-dependent kinases CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and found that CDK4/6 inhibitors destabilize HIF-1α under normoxia and hypoxia. To further investigate the molecular mechanism of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1α from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). We also identified a HIF-1α peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of SMURF2 increased basal expression of HIF-1α even in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1α and enhanced HIF-1α ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1α expression when Smurf2 was overexpressed. Smurf2 overexpression also inhibited HIF-1α expression level in two other cell lines, SW480 and VHL-deficient RCC4. Overexpression of SMURF2 mRNA is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation.

A novel 111indium-labeled dual carbonic anhydrase 9-targeted probe as a potential SPECT imaging radiotracer for detection of hypoxic colorectal cancer cells

Tumor hypoxia is a common feature in colorectal cancer (CRC), and is associated with resistance to radiotherapy and chemotherapy. Thus, a specifically targeted probe for the detection of hypoxic CRC cells is urgently needed. Carbonic anhydrase 9 (CA9) is considered to be a specific marker for hypoxic CRC diagnosis. Here, a nuclear imaging Indium-111 (111In)-labeled dual CA9-targeted probe was synthesized and evaluated for CA9 detection in in vitro, in vivo, and in human samples. The CA9-targeted peptide (CA9tp) and CA9 inhibitor acetazolamide (AAZ) were combined to form a dual CA9-targeted probe (AAZ-CA9tp) using an automatic microwave peptide synthesizer, which then was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radioisotope (111In) labeling (111In-DOTA-AAZ-CA9tp). The assays for cell binding, stability, and toxicity were conducted in hypoxic CRC HCT15 cells. The analyses for imaging and biodistribution were performed in an HCT15 xenograft mouse model. The binding and distribution of 111In-DOTA-AAZ-CA9tp were detected in human CRC samples using microautoradiography. AAZ-CA9tp possessed good CA9-targeting ability in hypoxic HCT15 cells. The dual CA9-targeted radiotracer showed high serum stability, high surface binding, and high affinity in vitro. After exposure of 111In-DOTA-AAZ-CA9tp to the HCT15-bearing xenograft mice, the levels of 111In-DOTA-AAZ-CA9tp were markedly and specifically increased in the hypoxic tumor tissues compared to control mice. 111In-DOTA-AAZ-CA9tp also targeted the areas of CA9 overexpression in human colorectal tumor tissue sections. The results of this study suggest that the novel 111In-DOTA-AAZ-CA9tp nuclear imaging agent may be a useful tool for the detection of hypoxic CRC cells in clinical practice.

PO-1928 Radiation cooperates with modulation of serine in human hypoxic colorectal cancer cells.

PO-1928 Radiation cooperates with modulation of serine in human hypoxic colorectal cancer cells.

Abstract 2014: Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase

Abstract The major adaptive response to hypoxia involves HIF-1α which is conventionally regulated by VHL E3 ligase. Our previous experiments suggested a stabilization of HIF-1α by CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and that CDK4/6 inhibitors destabilizes HIF-1α under normoxia and hypoxia. To further investigate the molecular mechanism of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1α from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). In addition, we identified a HIF-1α peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of Smurf2 increased basal expression of HIF-1α that was further increased in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1α and enhanced HIF-1α ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1α expression when Smurf2 was overexpressed. Overexpression of Smurf2 is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation. Citation Format: Shuai Zhao, Wafik S. El-Deiry. Identification of Smurf2 as a HIF-1α degrading E3 ubiquitin ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2014.

Hypoxic colorectal cancer-secreted exosomes deliver miR-210-3p to normoxic tumor cells to elicit a protumoral effect.

Hypoxia, the most common feature in the tumor microenvironment, is closely related to tumor malignant progression and poor patient's prognosis. Exosomes, initially recognized as cellular "garbage dumpsters", are now known to be important mediums for mediating cellular communication in tumor microenvironment. However, the mechanisms of hypoxic tumor cell-derived exosomes facilitate colorectal cancer progression still need further exploration. In the present study, we found that exosomes from hypoxic colorectal cancer cells (H-Exos) promoted G1-S cycle transition and proliferation while preventing the apoptosis of colorectal cancer cells by transmitting miR-210-3p to normoxic tumor cells. Mechanistic investigation indicated that miR-210-3p from H-Exos elicited its protumoral effect via suppressing CELF2 expression. A preclinical study further confirmed that H-Exos could promote tumorigenesis in vivo. Clinically, the expression of miR-210-3p in circulating plasma exosomes was markedly upregulated in colorectal cancer patients, which were closely associated with multiple unfavorable clinicopathological features. Taken together, these results suggest that hypoxia may stimulate colorectal cancer cells to secrete miR-210-3p-enriched exosomes in tumor microenvironment, which elicit protumoral effects by inhibiting CELF2 expression. These findings provide new insights on the mechanism of colorectal cancer progression and potential therapeutic targets for colorectal cancer.

Prion Protein of Extracellular Vesicle Regulates the Progression of Colorectal Cancer.

Simple SummaryCellular prion protein (PrPC) are overexpressed in cancers and related to cancer proliferation, invasion, metastasis, and drug resistance. The aim of our study was to investigate the role of PrPC-expressing exosomes regulating the colorectal cancer cells (CRC) behavior and tumor progression. We confirmed the increased sphere formation, expression of cancer initiating genes, motility, and tumor growth by hypoxic exosomes. Also, PrPC-expressing exosomes induced the microenvironment of metastasis via increase of endothelial permeability and angiogenic cytokine secretion. The treatment of anti-PrPC and 5-fluorouracil decreased the tumor progression. Targeting PrPC is an effective therapeutic strategy in cancer therapy.Colorectal cancer (CRC) is one of the leading causes of cancer-related death due to its aggressive metastasis in later stages. Although there is a growing interest in the tumorigenic role of cellular prion protein (PrPC) in the process of metastasis, the precise mechanism behind the cellular communication involving prion proteins remains poorly understood. This study found that hypoxic tumor microenvironment increased the PrPC-expressing exosomes from CRC, and these exosomes regulate the CRC cell behavior and tumor progression depending on the expression of PrPC. Hypoxic exosomes from CRC cells promoted sphere formation, the expression of tumor-inducing genes, migration, invasion, and tumor growth. Furthermore, these exosomes increased endothelial permeability, migration, invasion, and angiogenic cytokine secretion. These effects were associated with PrPC expression. Application of anti-PrPC antibody with 5-fluorouracil significantly suppressed the CRC progression in a murine xenograft model. Taken together, these findings indicate that PrP-expressing exosomes secreted by hypoxic CRC cells are a key factor in the tumorigenic CRC-to-CRC and CRC-to-endothelial cell communication. Significance: These findings suggest that inhibiting PrPC in hypoxic exosomes during chemotherapy may be an effective therapeutic strategy in colorectal cancer.

Hypoxia-induced Nur77 activates PI3K/Akt signaling via suppression of Dicer/let-7i-5p to induce epithelial-to-mesenchymal transition.

Background: Colorectal cancer (CRC) and the associated metastatic lesions are reported to be hypoxic. Hypoxia is a common feature in the tumor microenvironment and a potent stimulant of CRC. We have identified a regulatory role of Nur77 on Akt activation to enhance β-catenin signaling essential for CRC progression under hypoxic conditions.Methods: The functional role of Nur77 in hypoxia-induced EMT was examined by scattering assays to monitor the morphologies of CRC cell lines under 1% O2. Sphere formation assays were performed to investigate whether Nur77 induced cancer stem cell-like properties in hypoxic CRC cells. The expression of various epithelial-to-mesenchymal transition (EMT) and stemness markers was analyzed by qPCR and Western blotting. Finally, Nur77 function and signaling in vivo was ascertained in subcutaneous tumor xenograft or liver metastasis model in nude mice using CRC cells stably transfected with appropriate constructs.Results: Herein, we show, for the first time, that Nur77 is a novel regulator of microRNA biogenesis that may underlie its significant tumor-promoting activities in CRC cells under hypoxia. Mechanistically, Nur77 interacted with the tumor suppressor protein p63, leading to the inhibition of p63-dependent transcription of Dicer, an important miRNA processor and subsequent decrease in the biogenesis of let-7i-5p which targeted the 3'UTR of p110α mRNA and regulated its stability. Knockdown of Nur77 or overexpression of let-7i-5p inhibited the tumor metastasis in vivo.Conclusion: Our data uncovered a novel mechanistic link connecting Nur77, Akt, and invasive properties of CRC in the hypoxic microenvironment.

Hypoxic colorectal cancer cells promote metastasis of normoxic cancer cells depending on IL-8/p65 signaling pathway

Tumor heterogeneity is an important feature of malignant tumors, and cell subpopulations may positively interact to facilitate tumor progression. Studies have shown that hypoxic cancer cells possess enhanced metastatic capacity. However, it is still unclear whether hypoxic cancer cells may promote the metastasis of normoxic cells, which have greater access to the blood circulation. When cocultured with hypoxic CRC cells or treated with hypoxic CRC cell-derived CM, normoxic CRC cells possessed increased metastatic capacity. Furthermore, hypoxic CRC cell-derived CM was enriched in interleukin 8. Hypoxic CRC cell-derived CM and recombinant human IL-8 both enhanced the metastatic capacity of normoxic cells by increasing the phosphorylation of p65 and then by inducing epithelial-mesenchymal transition. Knockdown of IL-8 in hypoxic CRC cells or the use of an anti-IL-8 antibody attenuated the CM- or rhIL-8-induced prometastatic capacity of normoxic CRC cells. Inhibition or knockdown of p65 abrogated IL-8-induced prometastatic effects. Most importantly, hypoxia-treated xenograft tumors enhanced the metastasis of normoxic CRC cells. Hypoxic CRC cell-derived IL-8 promotes the metastatic capacity of normoxic cells, and novel therapies targeting the positive interactions between hypoxic and normoxic cells should be developed.