Glycolysis Inhibition of Autophagy Drives Malignancy in Ovarian Cancer: Exacerbation by IL-6 and Attenuation by Resveratrol.

peer reviewedCancer cells drive the glycolytic process towards the fermentation of pyruvate into lactate even in the presence of oxygen and functioning mitochondria, a phenomenon known as the "Warburg effect". Although not energetically efficient, glycolysis allows the cancer cell to synthesize the metabolites needed for cell duplication. Autophagy, a macromolecular degradation process, limits cell mass accumulation and opposes to cell proliferation as well as to cell migration. Cancer cells corrupt cancer-associated fibroblasts to release pro-inflammatory cytokines, which in turn promote glycolysis and support the metastatic dissemination of cancer cells. In mimicking in vitro this condition, we show that IL-6 promotes ovarian cancer cell migration only in the presence of glycolysis. The nutraceutical resveratrol (RV) counteracts glucose uptake and metabolism, reduces the production of reactive oxygen species consequent to excessive glycolysis, rescues the mitochondrial functional activity, and stimulates autophagy. Consistently, the lack of glucose as well as its metabolically inert analogue 2-deoxy-D-glucose (2-DG), which inhibits hexokinase 2 (HK2), trigger autophagy through mTOR inhibition, and prevents IL-6-induced cell migration. Of clinical relevance, bioinformatic analysis of The Cancer Genome Atlas dataset revealed that ovarian cancer patients bearing mutated TP53 with low expression of glycolytic markers and IL-6 receptor, together with markers of active autophagy, display a longer overall survival and are more responsive to platinum therapy. Taken together, our findings demonstrate that RV can counteract IL-6-promoted ovarian cancer progression by rescuing glycolysis-mediated inhibition of autophagy and support the view that targeting Warburg metabolism can be an effective strategy to limit the risk for cancer metastasis

Autophagy in Colorectal Cancer: Protective and Curative Effects of Probiotics

Autophagy is a catabolic lysosome-driven process crucial for the maintenance of cellular homeostasis. Any perturbation of this mechanism may dysregulate immune responses and impair bacteria clearance, causing chronic inflammation and favoring the dysbiotic alteration of the microbic flora. Gut dysbiosis is strongly associated with gastrointestinal inflammatory disorders that expose people to the risk of developing colorectal cancer (CRC) compared to healthy people. Recent studies have described the role of host microbiome in cancer initiation and progression, as well as in the modulation of therapeutic responses. The eubiotic state of gut microbiota can be restored by using dietary modulations (e.g., probiotics). Of relevance, in cancer cells there is a crucial interplay between the canonical Wnt pathway, whose regulation is strongly impaired by dysbiosis bacteria, and autophagy: B-Catenin, the key effector of the pathway, inhibits autophagy, while it is degraded by starvation-induced autophagy. Here, we explored the role of autophagy as mechanisms underlying the effects of the microbic flora in the restoration of intestinal homeostasis. We tested the metabolite butyrate on human CRC cell lines, and we examined the fate of the transcription factor B-Catenin. We demonstrated that butyrate-induced autophagy counteracted CRC cell proliferation and migration, promoting the autophagy degradation of B- Catenin. We proposed autophagy as alternative mechanisms for B-Catenin turn over in cancer cells, deficient for the B-Catenin degradation via proteosome. Moreover, we reported that the cell-free supernatant of the probiotic strain Lactiplantibacillus plantarum OC01 contrasted the malignant phenotypes of CRC cells induced by the inflammatory microenvironment. Taken together, we provided the preclinical rationale for the potential therapeutic strategies based on the modulation of microbiota. Manipulating intestinal bacteria may be a significant weapon in clinical practice.Autophagy is a catabolic lysosome-driven process crucial for the maintenance of cellular homeostasis. Any perturbation of this mechanism may dysregulate immune responses and impair bacteria clearance, causing chronic inflammation and favoring the dysbiotic alteration of the microbic flora. Gut dysbiosis is strongly associated with gastrointestinal inflammatory disorders that expose people to the risk of developing colorectal cancer (CRC) compared to healthy people. Recent studies have described the role of host microbiome in cancer initiation and progression, as well as in the modulation of therapeutic responses. The eubiotic state of gut microbiota can be restored by using dietary modulations (e.g., probiotics). Of relevance, in cancer cells there is a crucial interplay between the canonical Wnt pathway, whose regulation is strongly impaired by dysbiosis bacteria, and autophagy: B-Catenin, the key effector of the pathway, inhibits autophagy, while it is degraded by starvation-induced autophagy. Here, we explored the role of autophagy as mechanisms underlying the effects of the microbic flora in the restoration of intestinal homeostasis. We tested the metabolite butyrate on human CRC cell lines, and we examined the fate of the transcription factor B-Catenin. We demonstrated that butyrate-induced autophagy counteracted CRC cell proliferation and migration, promoting the autophagy degradation of B- Catenin. We proposed autophagy as alternative mechanisms for B-Catenin turn over in cancer cells, deficient for the B-Catenin degradation via proteosome. Moreover, we reported that the cell-free supernatant of the probiotic strain Lactiplantibacillus plantarum OC01 contrasted the malignant phenotypes of CRC cells induced by the inflammatory microenvironment. Taken together, we provided the preclinical rationale for the potential therapeutic strategies based on the modulation of microbiota. Manipulating intestinal bacteria may be a significant weapon in clinical practice.Autophagy is a catabolic lysosome-driven process crucial for the maintenance of cellular homeostasis. Any perturbation of this mechanism may dysregulate immune responses and impair bacteria clearance, causing chronic inflammation and favoring the dysbiotic alteration of the microbic flora. Gut dysbiosis is strongly associated with gastrointestinal inflammatory disorders that expose people to the risk of developing colorectal cancer (CRC) compared to healthy people. Recent studies have described the role of host microbiome in cancer initiation and progression, as well as in the modulation of therapeutic responses. The eubiotic state of gut microbiota can be restored by using dietary modulations (e.g., probiotics). Of relevance, in cancer cells there is a crucial interplay between the canonical Wnt pathway, whose regulation is strongly impaired by dysbiosis bacteria, and autophagy: B-Catenin, the key effector of the pathway, inhibits autophagy, while it is degraded by starvation-induced autophagy. Here, we explored the role of autophagy as mechanisms underlying the effects of the microbic flora in the restoration of intestinal homeostasis. We tested the metabolite butyrate on human CRC cell lines, and we examined the fate of the transcription factor B-Catenin. We demonstrated that butyrate-induced autophagy counteracted CRC cell proliferation and migration, promoting the autophagy degradation of B- Catenin. We proposed autophagy as alternative mechanisms for B-Catenin turn over in cancer cells, deficient for the B-Catenin degradation via proteosome. Moreover, we reported that the cell-free supernatant of the probiotic strain Lactiplantibacillus plantarum OC01 contrasted the malignant phenotypes of CRC cells induced by the inflammatory microenvironment. Taken together, we provided the preclinical rationale for the potential therapeutic strategies based on the modulation of microbiota. Manipulating intestinal bacteria may be a significant weapon in clinical practice

Ovarian Cancer Cell-Conditioning Medium Induces Cancer-Associated Fibroblast Phenoconversion through Glucose-Dependent Inhibition of Autophagy

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor–stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer

Cell-free Lactiplantibacillus plantarum OC01 supernatant suppresses IL-6-induced proliferation and invasion of human colorectal cancer cells: Effect on β-Catenin degradation and induction of autophagy

Background and aim: Gut microbiota is considered as a complex organ of human body. The interaction between the host and microbiota is dynamic and controlled by a huge number of factors, such as lifestyle, geography, pharmaceuticals, diet, and stress. The breakdown of this relationship could change microbiota composition favoring the onset of several diseases, including cancer. Metabolites released by microbiota bacterial strains have been reported to elicit protective effects on the mucosa that could contrast cancer development and progression. Here, we tested the ability of specific probiotic strain Lactiplantibacillus plantarum OC01-derived metabolites (NCIMB 30624) to contrast the malignant features of colorectal cancer (CRC) cells. Experimental procedure: The study was performed on two cell lines, HCT116 and HT29, cultured in 2D and 3D, and focused on the hallmarks of cell proliferation and migration. Results and conclusion: Probiotic metabolites reduced cell proliferation both in 2D and 3D-spheroid cultures, the latter model mimicking the growth in vivo. The bacterial metabolites also contrasted the pro-growth and pro-migratory activity of inteurleukin-6 (IL-6), an inflammatory cytokine abundantly found in the tumor microenvironment of CRC. These effects were associated with inhibition of the ERIC and of the mTOR/p70S6k pathways and with the inhibition of the E-to N-Cadherin switch. In a parallel study, we found that sodium butyrate (a representative of the main probiotic metabolites) induced autophagy and b-Catenin degradation, which is consistent with the growth inhibitory activity. The present data indicate that the metabolites of Lactiplantibacillus plantarum OC01 (NCIMB 30624) elicits anti-tumor effect and support its possible inclusion as adjuvant therapy of CRC for limiting cancer growth and progression. (c) 2023 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/)

BECN1 and BRCA1 Deficiency Sensitizes Ovarian Cancer to Platinum Therapy and Confers Better Prognosis

Background: BRCA1, BECN1 and TP53 are three tumor suppressor genes located on chromosome 17 and frequently found deleted, silenced, or mutated in many cancers. These genes are involved in autophagy, apoptosis, and drug resistance in ovarian cancer. Haploinsufficiency or loss-of-function of either TP53, BRCA1 or BECN1 correlates with enhanced predisposition to cancer development and progression, and chemoresistance. Expectedly, the combined altered expression of these three tumor suppressor genes worsens the prognosis of ovarian cancer patients. However, whether such a genotypic pattern indeed affects the chemo-responsiveness to standard chemotherapy thus worsening patients' survival has not been validated in a large cohort of ovarian cancer patients. Aim: We interrogated datasets from the TCGA database to analyze how the expression of these three tumor suppressor genes impacts on the clinical response to platinum-based chemotherapy thus affecting the survival of ovarian cancer patients. Results and conclusion: Compared to EOC with homozygous expression of BECN1 and BRCA1, tumors expressing low mRNA expression of these two tumor suppressor genes (either because of shallow (monoallelic) co-deletion or of promoter hypermethylation), showed higher sensitivity to platinum-based therapies and were associated with a better prognosis of ovarian cancer-bearing patients. This outcome was independent of TP53 status, though it was statistically more significant in the cohort of patients with mutated TP53. Thus, sensitivity to platinum therapy (and probably to other chemotherapeutics) correlates with low expression of a combination of critical tumor suppressor genes. Our study highlights the importance of thoroughly assessing the genetic lesions of the most frequently mutated genes to stratify the patients in view of a personalized therapy. More importantly, the present findings suggest that targeting the function of both BECN1 and BRCA1 could be a strategy to restore chemosensitivity in refractory tumors

Butyrate Inhibits Colorectal Cancer Cell Proliferation through Autophagy Degradation of β-Catenin Regardless of APC and β-Catenin Mutational Status

Colorectal cancer (CRC) pathogenesis is mainly driven by alterations in WNT signaling, which results in altered transcriptional activity of β-Catenin. Mutations in APC (Adenomatous Polyposis Coli) are reflected in β-Catenin hyperactivation and loss of proliferation control. Certain intestinal bacteria metabolites have shown the ability to limit CRC cell proliferation and CRC pathogenesis. Here, we investigated the molecular mechanism underlying the anti-proliferative activity of butyrate, a microbiota-derived short chain fatty acid, in two CRC cell lines, namely HCT116 and SW620, which bear a mutation in β-Catenin and APC, respectively. In particular, we focused on autophagy, a lysosome-dependent degradation pathway, which was shown to control intestinal tissue homeostasis. Butyrate reduced CRC cell proliferation, as witnessed by the downregulation of proliferation markers. TCGA bioinformatic transcriptomic analysis of CTNNB1 (β-Catenin) gene correlation in CRC patients showed that β-Catenin negatively correlates with the autophagy gene ATG4D. In CRC cells, regardless of the mutational state of APC or β-Catenin genes, butyrate caused the autophagy-mediated degradation of β-Catenin; thus, preventing its transcriptional activity. Autophagy gene silencing restored β-Catenin levels, allowing it to translocate into the nucleus to promote the expression of downstream genes associated with cancer cell proliferation. CRC-affected patients show driver mutations in the WNT pathway; thus, targeting its crucial effector may be a promising therapeutic strategy in CRC treatment; for instance, by using ad hoc probiotics that stimulate autophagy

Differential Competitive Growth of Transgenic Subclones of Neuroblastoma Cells Expressing Different Levels of Cathepsin D Co-Cultured in 2D and 3D in Response to EGF: Implications in Tumor Heterogeneity and Metastasis

Neuroblastoma (NB) is an embryonal tumor arising from the sympathetic central nervous system. The epidermal growth factor (EGF) plays a role in NB growth and metastatic behavior. Recently, we have demonstrated that cathepsin D (CD) contrasts EGF-induced NB cell growth in 2D by downregulating EGFR/MAPK signaling. Aggressive NB is highly metastatic to the bone and the brain. In the metastatic process, adherent cells detach to form clusters of suspended cells that adhere once they reach the metastatic site and form secondary colonies. Whether CD is involved in the survival of metastatic NB clones is not known. Therefore, in this study, we addressed how CD differentially affects cell growth in suspension versus the adherent condition. To mimic tumor heterogeneity, we co-cultured transgenic clones silenced for or overexpressing CD. We compared the growth kinetics of such mixed clones in 2D and 3D models in response to EGF, and we found that the Over CD clone had an advantage for growth in suspension, while the CD knocked-down clone was favored for the adherent growth in 2D. Interestingly, on switching from 3D to 2D culture conditions, the expression of E-cadherin and of N-cadherin increased in the KD-CD and Over CD clones, respectively. The fact that CD plays a dual role in cancer cell growth in 2D and 3D conditions indicates that during clonal evolution, subclones expressing different level of CD may arise, which confers survival and growth advantages depending on the metastatic step. By searching the TCGA database, we found up to 38 miRNAs capable of downregulating CD. Interestingly, these miRNAs are associated with biological processes controlling cell adhesion and cell migration. The present findings support the view that during NB growth on a substrate or when spreading as floating neurospheres, CD expression is epigenetically modulated to confer survival advantage. Thus, epigenetic targeting of CD could represent an additional strategy to prevent NB metastases

High BECN1 Expression Negatively Correlates with BCL2 Expression and Predicts Better Prognosis in Diffuse Large B-Cell Lymphoma: Role of Autophagy

Diffuse large B-cell lymphoma (DLBCL) is characterized by high molecular and clinical heterogeneity. Autophagy, a lysosome-driven catabolic process devoted to macromolecular turnover, is fundamental in maintaining normal hematopoietic stem cells and progenitors homeostasis, and its dysregulation plays a critical role in the initiation and progression of hematological malignancies. One main regulator of autophagy is BECLIN-1, which may interact alternatively with either BCL-2, thus allowing apoptosis, or PI3KC3, thus promoting autophagy. The altered expression of BCL2 and BECN1 correlates with lymphoma outcomes, but whether this is associated with dysregulated cross-talk between autophagy and apoptosis remains to be elucidated. Analysis of the TCGA database revealed that BCL2 and BECN1 mRNA expression were inversely correlated in DLBCL patients. In representative DLBCL cell lines exposed to doxorubicin, the cells highly expressing BCL-2 were resistant, while the ones highly expressing BECLIN-1 were sensitive, and this correlated with low and high autophagy flux, respectively. Venetoclax targeting of BCL-2 increased while the spautin-1-mediated inhibition of BECLIN-1-dependent autophagy reversed doxorubicin sensitivity in the former and in the latter, respectively. By interrogating the TCGA DLBCL dataset, we found that BCL2 and BECN1 acted as negative and positive prognostic markers for DLBCL, respectively. The differentially expressed gene analysis in the respective cohorts revealed that BCL2 positively correlated with oncogenic pathways (e.g., glucose transport, HIF1A signaling, JAK-STAT signaling, PI3K-AKT-mTOR pathway) and negatively correlated with autophagy-related transcripts, while BECN1 showed the opposite trend. Notably, patients with high BECN1 expression displayed longer survival. Our data reveal, for the first time, that the modulation of BECLIN-1-dependent autophagy influences the prognosis of DLBCL patients and provide a mechanistic explanation supporting the therapeutic use of drugs that, by stimulating autophagy, can sensitize lymphoma cells to chemotherapy

Preclinical evidence for preventive and curative effects of resveratrol on xenograft cholangiocarcinogenesis

Cholangiocarcinoma (CCA), the malignant tumor of bile duct epithelial cells, is a relatively rare yet highly lethal cancer. In this work, we tested the ability of Resveratrol (RV) to prevent and cure CCA xenograft in nude mice and investigated molecular mechanisms underpinning such anticancer effect. Human CCA cells were xenografted in mice that were or not treated prior to or after to transplantation with RV. Tumor growth was monitored and analyzed for the markers of cell proliferation, apoptosis, and autophagy. TCGA was interrogated for the molecules possibly targeted by RV. RV could inhibit the growth of human CCA xenograft when administered after implantation and could reduce the growth or even impair the implantation of the tumors when administered prior the transplantation. RV inhibited CCA cell proliferation, induced apoptosis with autophagy, and strongly reduced the presence of CAFs and production of IL-6. Interrogation of CCA dataset in TCGA database revealed that the expression of IL-6 Receptor (IL-6R) inversely correlated with that of MAP-LC3 and BECLIN-1, and that low expression of IL-6R and of MIK67, two pathways downregulated by RV, associated with better survival of CCA patients. Our data demonstrate that RV elicits a strong preventive and curative anticancer effect in CCA by limiting the formation of CAFs and their release of IL-6, and this results in up-regulation of autophagy and apoptosis in the cancer cells. These findings support the clinical use of RV as a primary line of prevention in patients exposed at risk and as an adjuvant therapeutics in CCA patients