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.

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.

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.

Cell-free Lactiplantibacillus plantarum OC01 supernatant suppresses IL-6-induced proliferation and invasion of human colorectal cancer cells: Effect on ß-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 ERK 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 ß-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.

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

Cancer 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.

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

Colorectal cancer (CRC) pathogenesis is mainly driven by alterations in WNT signaling, which results in altered transcriptional activity of ß-Catenin. Mutations in APC (Adenomatous Polyposis Coli) are reflected in ß-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 ß-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 (ß-Catenin) gene correlation in CRC patients showed that ß-Catenin negatively correlates with the autophagy gene ATG4D. In CRC cells, regardless of the mutational state of APC or ß-Catenin genes, butyrate caused the autophagy-mediated degradation of ß-Catenin; thus, preventing its transcriptional activity. Autophagy gene silencing restored ß-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.

Resveratrol Contrasts LPA-Induced Ovarian Cancer Cell Migration and Platinum Resistance by Rescuing Hedgehog-Mediated Autophagy.

Background: Ovarian cancer progression and invasiveness are promoted by a range of soluble factors released by cancer cells and stromal cells within the tumor microenvironment. Our previous studies demonstrated that resveratrol (RV), a nutraceutical and caloric restriction mimetic with tumor-suppressive properties, counteracts cancer cell motility induced by stromal IL-6 by upregulating autophagy. Lysophosphatidic acid (LPA), a bioactive phospholipid that shows elevated levels in the tumor microenvironment and the ascites of ovarian cancers, stimulates the growth and tissue invasion of cancer cells. Whether LPA elicits these effects by inhibiting autophagy and through which pathway and whether RV can counteract the same remain obscure. Aims: To investigate the molecular pathways involved in LPA-induced ovarian cancer malignancy, particularly focusing on the role of autophagy, and the ability of RV to counteract LPA activity. Results: LPA stimulated while RV inhibited ovarian cancer cell migration. Transcriptomic and bioinformatic analyses showed an opposite regulation by LPA and RV of genes linked to epithelial-to-mesenchymal transition (EMT) and autophagy with involvement of the PI3K-AKT, JAK-STAT and Hedgehog (Hh) pathways. LPA upregulated the Hh and EMT members GLI1, BMI-1, SNAIL-1 and TWIST1 and inhibited autophagy, while RV did the opposite. Similar to the inhibitors of the Hh pathway, RV inhibited LPA-induced cancer cell migration and 3D growth of ovarian cancer cells. BMI-1 silencing prevented LPA-induced EMT, restored autophagy and hampered cell migration, resembling the effects of RV. TCGA data analyses indicated that patients with low expression of Hh/EMT-related genes together with active autophagy flux tended to have a better prognosis and this correlates with a more effective response to platinum therapy. In in vitro 3D spheroids, LPA upregulated BMI-1, downregulated autophagy and inhibited platinum toxicity while RV and Hh inhibitors restored autophagy and favored BAX-mediated cell death in response to platinum. Conclusions: By inhibiting the Hh pathway and restoration of autophagy, RV counteracts LPA-induced malignancy, supporting its inclusion in the therapy of ovarian cancer for limiting metastasis and chemoresistance.

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.

How Autophagy Shapes the Tumor Microenvironment in Ovarian Cancer.

Ovarian cancer (OC) is characterized by a high mortality rate due to the late diagnosis and the elevated metastatic potential. Autophagy, a lysosomal-driven catabolic process, contributes to the macromolecular turnover, cell homeostasis, and survival, and as such, it represents a pathway targetable for anti-cancer therapies. It is now recognized that the vascularization and the cellular composition of the tumor microenvironment influence the development and progression of OC by controlling the availability of nutrients, oxygen, growth factors, and inflammatory and immune-regulatory soluble factors that ultimately impinge on autophagy regulation in cancer cells. An increasing body of evidence indicates that OC carcinogenesis is associated, at least in the early stages, to insufficient autophagy. On the other hand, when the tumor is already established, autophagy activation provides a survival advantage to the cancer cells that face metabolic stress and protects from the macromolecules and organelles damages induced by chemo- and radiotherapy. Additionally, upregulation of autophagy may lead cancer cells to a non-proliferative dormant state that protects the cells from toxic injuries while preserving their stem-like properties. Further to complicate the picture, autophagy is deregulated also in stromal cells. Thus, changes in the tumor microenvironment reflect on the metabolic crosstalk between cancer and stromal cells impacting on their autophagy levels and, consequently, on cancer progression. Here, we present a brief overview of the role of autophagy in OC hallmarks, including tumor dormancy, chemoresistance, metastasis, and cell metabolism, with an emphasis on the bidirectional metabolic crosstalk between cancer cells and stromal cells in shaping the OC microenvironment.

Modulation of non-coding RNAs by resveratrol in ovarian cancer cells: In silico analysis and literature review of the anti-cancer pathways involved.

BACKGROUND AND AIM: Non-coding RNAs control cell functioning through affecting gene expression and translation and their dysregulation is associated with altered cell homeostasis and diseases, including cancer. Nutraceuticals with anti-cancer therapeutic potential have been shown to modulate non-coding RNAs expression that could impact on the expression of genes involved in the malignant phenotype. EXPERIMENTAL PROCEDURE: Here, we report on the microarray profiling of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) and on the associated biochemical pathways and functional processes potentially modulated in OVCAR-3 ovarian cancer cells exposed for 24 h to Resveratrol (RV), a nutraceutical that has been shown to inhibit carcinogenesis and cancer progression in a variety of human and animal models, both in vitro and in vivo. Diana tools and Gene Ontology (GO) pathway analyses along with Pubmed literature search were employed to identify the cellular processes possibly affected by the dysregulated miRNAs and lncRNAs. RESULTS AND CONCLUSION: The present data consistently support the contention that RV could exert anti-neoplastic activity via non-coding RNAs epigenetic modulation of the pathways governing cell homeostasis, cell proliferation, cell death and cell motility.

Epigenetic targeting of autophagy for cancer prevention and treatment by natural compounds.

Despite the undeniable progress made in the last decades, cancer continues to challenge the scientists engaged in searching for an effective treatment for its prevention and cure. One of the malignant hallmarks that characterize cancer cell biology is the altered metabolism of sugars and amino acids. Autophagy is a pathway allowing the macromolecular turnover via recycling of the substrates resulting from the lysosomal degradation of damaged or redundant cell molecules and organelles. As such, autophagy guarantees the proteome quality control and cell homeostasis. Data from in vitro, in animals and in patients researches show that dysregulation of autophagy favors carcinogenesis and cancer progression, making this process an ineluctable target of cancer therapy. The autophagy process is regulated at genetic, epigenetic and post-translational levels. Targeting autophagy with epigenetic modifiers could represent a valuable strategy to prevent or treat cancer. A wealth of natural products from terrestrial and marine living organisms possess anti-cancer activity. Here, we review the experimental proofs demonstrating the ability of natural compounds to regulate autophagy in cancer via epigenetics. The hope is that in the near future this knowledge could translate into effective intervention to prevent and cure cancer.

Autophagy drives osteogenic differentiation of human gingival mesenchymal stem cells.

BACKGROUND/AIM: Autophagy is a macromolecular degradation process playing a pivotal role in the maintenance of stem-like features and in the morpho-functional remodeling of the tissues undergoing differentiation. In this work we investigated the involvement of autophagy in the osteogenic differentiation of mesenchymal stem cells originated from human gingiva (HGMSC). METHODS: To promote the osteogenic differentiation of HGMSCs we employed resveratrol, a nutraceutical known to modulate autophagy and cell differentiation, together with osteoblastic inductive factors. Osteoblastic differentiation and autophagy were monitored through western blotting and immunofluorescence staining of specific markers. RESULTS: We show that HGMSCs can differentiate into osteoblasts when cultured in the presence of appropriate factors and that resveratrol accelerates this process by up-regulating autophagy. The prolonged incubation with dexamethasone, ß-glycerophosphate and ascorbic acid induced the osteogenic differentiation of HGMSCc with increased expression of autophagy markers. Resveratrol (1 µM) alone elicited a less marked osteogenic differentiation yet it greatly induced autophagy and, when added to the osteogenic differentiation factors, it provoked a synergistic effect. Resveratrol and osteogenic inductive factors synergistically induced the AMPK-BECLIN-1 pro-autophagic pathway in differentiating HGMSCs, that was thereafter downregulated in osteoblastic differentiated cells. Pharmacologic inhibition of BECLIN-1-dependent autophagy precluded the osteogenic differentiation of HGMSCs. CONCLUSIONS: Autophagy modulation is instrumental for osteoblastic differentiation of HGMSCs. The present findings can be translated into the regenerative cell therapy of maxillary / mandibular bone defects.