Metformin and Breast Cancer: Where Are We Now?

Breast cancer is the most prevalent cancer and the leading cause of cancer-related death among women worldwide. Type 2 diabetes-associated metabolic traits such as hyperglycemia, hyperinsulinemia, inflammation, oxidative stress, and obesity are well-known risk factors for breast cancer. The insulin sensitizer metformin, one of the most prescribed oral antidiabetic drugs, has been suggested to function as an antitumoral agent, based on epidemiological and retrospective clinical data as well as preclinical studies showing an antiproliferative effect in cultured breast cancer cells and animal models. These benefits provided a strong rationale to study the effects of metformin in routine clinical care of breast cancer patients. However, the initial enthusiasm was tempered after disappointing results in randomized controlled trials, particularly in the metastatic setting. Here, we revisit the current state of the art of metformin mechanisms of action, critically review past and current metformin-based clinical trials, and briefly discuss future perspectives on how to incorporate metformin into the oncologist's armamentarium for the prevention and treatment of breast cancer.

An olive oil phenolic is a new chemotype of mutant isocitrate dehydrogenase 1 (IDH1) inhibitors.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene confer an oncogenic gain-of-function activity that allows the conversion of α-ketoglutarate (α-KG) to the oncometabolite R-2-hydroxyglutarate (2HG). The accumulation of 2HG inhibits α-KG-dependent histone and DNA demethylases, thereby generating genome-wide hypermethylation phenotypes with cancer-initiating properties. Several chemotypes of mutant IDH1/2-targeted inhibitors have been reported, and some of them are under evaluation in clinical trials. However, the recognition of acquired resistance to such inhibitors within a few years of clinical use raises an urgent need to discover new mutant IDH1 antagonists. Here, we report that a naturally occurring phenolic compound in extra-virgin olive oil (EVOO) selectively inhibits the production of 2HG by neomorphic IDH1 mutations. In silico docking, molecular dynamics, including steered simulations, predicted the ability of the oleoside decarboxymethyl oleuropein aglycone (DOA) to preferentially occupy the allosteric pocket of mutant IDH1. DOA inhibited the enzymatic activity of recombinant mutant IDH1 (R132H) protein in the low micromolar range, whereas >10-fold higher concentrations were required to inhibit the activity of wild-type (WT) IDH1. DOA suppressed 2HG overproduction in engineered human cells expressing a heterozygous IDH1-R132H mutation. DOA restored the 2HG-suppressed activity of histone demethylases as it fully reversed the hypermethylation of H3K9me3 in IDH1-mutant cells. DOA epigenetically restored the expression of PD-L1, an immunosuppressive gene silenced in IDH1 mutant cells via 2HG-driven DNA hypermethylation. DOA selectively blocked colony formation of IDH1 mutant cells while sparing WT IDH1 isogenic counterparts. In sum, the EVOO-derived oleoside DOA is a new, naturally occurring chemotype of mutant IDH1 inhibitors.

Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells.

Targeting tumor-initiating, drug-resistant populations of cancer stem cells (CSC) with phytochemicals is a novel paradigm for cancer prevention and treatment. We herein employed a phenotypic drug discovery approach coupled to mechanism-of-action profiling and target deconvolution to identify phenolic components of extra virgin olive oil (EVOO) capable of suppressing the functional traits of CSC in breast cancer (BC). In vitro screening revealed that the secoiridoid decarboxymethyl oleuropein aglycone (DOA) could selectively target subpopulations of epithelial-like, aldehyde dehydrogenase (ALDH)-positive and mesenchymal-like, CD44+CD24-/low CSC. DOA could potently block the formation of multicellular tumorspheres generated from single-founder stem-like cells in a panel of genetically diverse BC models. Pretreatment of BC populations with noncytotoxic doses of DOA dramatically reduced subsequent tumor-forming capacity in vivo. Mice orthotopically injected with CSC-enriched BC-cell populations pretreated with DOA remained tumor-free for several months. Phenotype microarray-based screening pointed to a synergistic interaction of DOA with the mTOR inhibitor rapamycin and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine. In silico computational studies indicated that DOA binds and inhibits the ATP-binding kinase domain site of mTOR and the S-adenosyl-l-methionine (SAM) cofactor-binding pocket of DNMTs. FRET-based Z-LYTE™ and AlphaScreen-based in vitro assays confirmed the ability of DOA to function as an ATP-competitive mTOR inhibitor and to block the SAM-dependent methylation activity of DNMTs. Our systematic in vitro, in vivo and in silico approaches establish the phenol-conjugated oleoside DOA as a dual mTOR/DNMT inhibitor naturally occurring in EVOO that functionally suppresses CSC-like states responsible for maintaining tumor-initiating cell properties within BC populations.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

Fatty acid synthase (FASN) signalome: A molecular guide for precision oncology.

The initial excitement generated more than two decades ago by the discovery of drugs targeting fatty acid synthase (FASN)-catalyzed de novo lipogenesis for cancer therapy was short-lived. However, the advent of the first clinical-grade FASN inhibitor (TVB-2640; denifanstat), which is currently being studied in various phase II trials, and the exciting advances in understanding the FASN signalome are fueling a renewed interest in FASN-targeted strategies for the treatment and prevention of cancer. Here, we provide a detailed overview of how FASN can drive phenotypic plasticity and cell fate decisions, mitochondrial regulation of cell death, immune escape and organ-specific metastatic potential. We then present a variety of FASN-targeted therapeutic approaches that address the major challenges facing FASN therapy. These include limitations of current FASN inhibitors and the lack of precision tools to maximize the therapeutic potential of FASN inhibitors in the clinic. Rethinking the role of FASN as a signal transducer in cancer pathogenesis may provide molecularly driven strategies to optimize FASN as a long-awaited target for cancer therapeutics.

Evolution of the predictive markers amphiregulin and epiregulin mRNAs during long-term cetuximab treatment of KRAS wild-type tumor cells.

Molecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX's molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions.

Ideal oncology drugs would be curative after a short treatment course if they could eliminate epithelium-originated carcinomas at their non-invasive, pre-malignant stages. Such ideal molecules, which are expected to molecularly abrogate all the instrumental mechanisms acquired by migrating cancer stem cells (CSCs) to by-pass tumour suppressor barriers, might already exist. We here illustrate how system biology strategies for repositioning existing FDA-approved drugs may accelerate our therapeutic capacity to eliminate CSC traits in pre-invasive intraepithelial neoplasias. First, we describe a signalling network signature that overrides bioenergetics stress- and oncogene-induced senescence (OIS) phenomena in CSCs residing at pre-invasive lesions. Second, we functionally map the anti-malarial chloroquine and the anti-diabetic metformin ("old drugs") to their recently recognized CSC targets ("new uses") within the network. By discussing the preclinical efficacy of chloroquine and metformin to inhibiting the genesis and self-renewal of CSCs we finally underscore the expected translational impact of the "old drugs-new uses" repurposing strategy to open a new CSC-targeted chemoprevention era.

Circulating levels of MOTS-c in patients with breast cancer treated with metformin.

The mitokine MOTS-c is a mitochondrially-encoded "exercise-mimetic peptide" expressed in multiple tissues, particularly skeletal muscles, which can be detected as a circulating hormone in the blood. MOTS-c mechanisms of action (MoA) involve insulin sensitization, enhanced glucose utilization, suppression of mitochondrial respiration, and targeting of the folate-AICAR-AMPK pathway. Although MOTS-c MoA largely overlap those of the anti-diabetic biguanide metformin, the putative regulatory actions of metformin on MOTS-c have not yet been evaluated in detail. Here, we measured circulating MOTS-c in paired baseline and post-treatment sera obtained from HER2-positive breast cancer patients randomized to receive either metformin combined with neoadjuvant chemotherapy and trastuzumab or an equivalent regimen without metformin. We failed to find any significant alteration of circulating MOTS-c -as measured using the commercially available competitive ELISA CEX132Hu- in response to 24 weeks of a neoadjuvant chemotherapy/trastuzumab regimen with or without daily metformin. Changes in circulating MOTS-c levels failed to reach statistical significance when comparing patients achieving pathological complete response (pCR), irrespective of metformin treatment. The inability of metformin to target skeletal muscle, the major tissue for MOTS-c production and secretion, might limit its regulatory effects on circulating MOTS-c. Further studies are needed to definitely elucidate the nature of the interaction between metformin and MOTS-c in cancer and non-cancer patients.

Lapatinib, a dual HER1/HER2 tyrosine kinase inhibitor, augments basal cleavage of HER2 extracellular domain (ECD) to inhibit HER2-driven cancer cell growth.

The ultimate biological and clinical meaning of shed HER2 extracellular domain (ECD) has remained largely unclear until recently. Oversecretion of soluble HER2 ECD has been shown to inhibit growth of HER2-overexpressing cancer cells by promoting HER2 ECD dimerization with HER transmembrane receptors thus impairing their cross-tyrosine phosphorylation and decreasing their activation status. HER2-targeted drugs capable to enhance the occurrence of basal HER2 ECD shedding but simultaneously preventing formation of truncated cell membrane-bound HER2 intracellular fragment, which exhibits an undesirable constitutive kinase activity, might be extremely efficient at managing HER2-positive cancer disease. The dual HER1/HER2 Tyrosine Kinase inhibitor lapatinib, which works intracellularly and directly targets the TK domain of HER2, drastically augments basal shedding of HER2 ECD to inhibit HER2-driven cancer cell growth. Lapatinib treatment significantly augments the concentration of the inactive (unphosphorylated) form of HER2 protein at the tumor cell membrane and promotes an exacerbated HER2 ECD shedding to the extracellular milieu of HER2-overexpressing cancer cells. Exacerbated sensitivity of trastuzumab-resistant cancer cells, which contain nearly undetectable levels of soluble HER2 ECD when compared with trastuzumab-sensitive parental cells to lapatinib-induced cell growth inhibition, takes place when lapatinib treatment fully restores high levels of basal HER2 ECD shedding. The dramatic augmentation of HER2 ECD shedding that occurs upon treatment of with lapatinib is fully suppressed in lapatinib-refractory HER2-positive cells. These findings, altogether, may provide crucial insights concerning clinical studies aimed to accurately describe HER2 ECD as a potential predictor of response or resistance to the HER2-targeted drugs trastuzumab and lapatinib.

Stem cell property epithelial-to-mesenchymal transition is a core transcriptional network for predicting cetuximab (Erbitux™) efficacy in KRAS wild-type tumor cells.

Beyond a well-recognized effect of KRAS mutations in determining de novo inefficacy of cetuximab (CTX) in metastatic colorectal cancer, we urgently need a biomarker signature for predicting CTX efficacy in KRAS wild-type (WT) tumors. CTX-adapted EGFR gene-amplified KRAS WT tumor cell populations were induced by stepwise-chronic exposure of A431 epidermoid cancer cells to CTX. Genome-wide analyses of 44K Agilent's whole human arrays were bioinformatically evaluated by Gene Set Enrichment Analysis (GSEA)-based screening of the KEGG pathway database. Molecular functioning of CTX was found to depend on: (i) The occurrence of a positive feedback loop on Epidermal Growth Factor Receptor (EGFR) activation driven by genes coding for EGFR ligands (e.g., amphiregulin); (ii) the lack of a negative feedback on mitogen-activated protein kinase (MAPK) activation regulated by dual-specificity phosphatases (e.g., DUSP6) and; (iii) the transcriptional status of gene pathways controlling the epithelial-to-mesenchymal transition (EMT) and its reversal (MET) program (actin cytoskeleton and cell-cell communication-e.g., keratins-focal adhesion signaling-e.g., integrins-and EMT-inducing cytokines - e.g., transforming growth factor-ß). Quantitative real-time PCR, high-content immunostaining, and flow-cytometry analyses confirmed that CTX efficacy depends on its ability to promote: (i) Stronger cell-cell contacts by up-regulating the expression of the epithelial markers E-cadherin and occludin; (ii) down-regulation of the epithelial transcriptional repressors Zeb, Snail, and Slug accompanied by restoration of cortical F-actin; and (iii) complete prevention of the CD44(pos)/CD24(neg/low) mesenchymal immunophenotype. The impact of EGFR ligands/MAPK phosphatases gene transcripts in predicting CTX efficacy in KRAS WT tumors may be tightly linked with the ability of CTX to concurrently reverse the EMT status, a pivotal property of migrating cancer stem cells.

Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2 gene-amplified breast cancer cells with primary resistance to HER1/2-targeted therapies.

Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC50 values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naïve SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed ~ 4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated ~10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely suppressed in JIMT-1 cells. Our current findings may be extremely helpful to design successful combinatorial strategies aimed to circumvent the occurrence of de novo resistance to HER2-directed drugs using survivin antagonists.

The anti-diabetic drug metformin suppresses self-renewal and proliferation of trastuzumab-resistant tumor-initiating breast cancer stem cells.

We here demonstrate that the anti-diabetic drug metformin interacts synergistically with the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin™) to eliminate stem/progenitor cell populations in HER2-gene-amplified breast carcinoma cells. When using the mammosphere culture technique, graded concentrations of single-agent metformin (range 50-1,000 µmol/l) were found to dose-dependently reduce the number of mammospheres formed by SKBR3 (a Tzb-naïve model), SKBR3 TzbR (a model of acquired auto-resistance to Tzb) and JIMT-1 (a model of refractoriness to Tzb and other HER2-targeted therapies ab initio) HER2-overexpressing breast cancer cells. Single-agent Tzb likewise reduced mammosphere-forming efficiency (MSFE) in Tzb-naïve SKBR3 cells, but it failed to significantly decrease MSFE in Tzb-resistant SKBR3 TzbR and JIMT-1 cells. Of note, CD44-overexpressing Tzb-refractory SKBR3 TzbR and JIMT-1 cells retained an exquisite sensitivity to single-agent metformin. Concurrent combination of metformin with Tzb synergistically reduced MSFE as well as the size of mammospheres in Tzb-refractory SKBR3 TzbR and JIMT-1 cells. Flow cytometry analyses confirmed that metformin and Tzb functioned synergistically to down-regulate the percentage of Tzb-refractory JIMT-1 cells displaying the CD44(pos)/CD24(neg/low) stem/progenitor immunophenotype. Given that MSFE and mammosphere size are indicators of stem self-renewal and progenitor cell proliferation, respectively, our current findings reveal for the first time that: (a) Tzb refractoriness in HER2 overexpressors can be explained in terms of Tzb-resistant/CD44-overexpressing/tumor-initiating stem cells; (b) metformin synergistically interacts with Tzb to suppress self-renewal and proliferation of cancer stem/progenitor cells in HER2-positive carcinomas.

Clinical Management of COVID-19 in Cancer Patients with the STAT3 Inhibitor Silibinin.

COVID-19 pathophysiology is caused by a cascade of respiratory and multiorgan failures arising, at least in part, from the SARS-CoV-2-driven dysregulation of the master transcriptional factor STAT3. Pharmacological correction of STAT3 over-stimulation, which is at the root of acute respiratory distress syndrome (ARDS) and coagulopathy/thrombosis events, should be considered for treatment of severe COVID-19. In this perspective, we first review the current body of knowledge on the role of STAT3 in the pathogenesis of severe COVID-19. We then exemplify the potential clinical value of treating COVID-19 disease with STAT3 inhibitors by presenting the outcomes of two hospitalized patients with active cancer and COVID-19 receiving oral Legalon®-a nutraceutical containing the naturally occurring STAT3 inhibitor silibinin. Both patients, which were recruited to the clinical trial SIL-COVID19 (EudraCT number: 2020-001794-77) had SARS-CoV-2 bilateral interstitial pneumonia and a high COVID-GRAM score, and showed systemic proinflammatory responses in terms of lymphocytopenia and hypoalbuminemia. Both patients were predicted to be at high risk of critical COVID-19 illness in terms of intensive care unit admission, invasive ventilation, or death. In addition to physician's choice of best available therapy or supportive care, patients received 1050 mg/day Legalon® for 10 days without side-effects. Silibinin-treated cancer/COVID-19+ patients required only minimal oxygen support (2-4 L/min) during the episode, exhibited a sharp decline of the STAT3-regulated C-reactive protein, and demonstrated complete resolution of the pulmonary lesions. These findings might inspire future research to advance our knowledge and improve silibinin-based clinical interventions aimed to target STAT3-driven COVID-19 pathophysiology.

Metformin Is a Pyridoxal-5'-phosphate (PLP)-Competitive Inhibitor of SHMT2.

The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5'-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.

Dynamic emergence of the mesenchymal CD44(pos)CD24(neg/low) phenotype in HER2-gene amplified breast cancer cells with de novo resistance to trastuzumab (Herceptin).

Evidence is mounting that the occurrence of the CD44(pos)/CD24(neg/low) cell population, which contains potential breast cancer (BC) stem cells, could explain BC clinical resistance to HER2-targeted therapies. We investigated whether de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin) may relate to the dynamic regulation of the mesenchymal CD44(pos)/CD24(neg/low) phenotype in HER2-positive BC. We observed that the subpopulation of Tzb-refractory JIMT-1 BC cells exhibiting CD44(pos)/CD24(neg/low)-surface markers switched with time. Low-passage JIMT-1 cell cultures were found to spontaneously contain approximately 10% of cells bearing the CD44(pos)/CD24(neg/low) immunophenotype. Late-passage (>60) JIMT-1 cultures accumulated approximately 80% of CD44(pos)/CD24(neg/low) cells and closely resembled the CD44(pos)/CD24(neg/low)-enriched ( approximately 85%) cell population constitutively occurring in HER2-negative MDA-MB-231 mesenchymal BC cells. Dynamic expression of mesenchymal markers was not limited to CD44/CD24 because high-passages of JIMT-1 cells exhibited also reduced expression of the HER2 protein and over-secretion of pro-invasive/metastatic chemokines and metalloproteases. Accordingly, late-passage JIMT-1 cells displayed an exacerbated migratogenic phenotype in plastic, collagen, and fibronectin substrates. Intrinsic genetic plasticity to efficiently drive the emergence of the CD44(pos)/CD24(neg/low) mesenchymal phenotype may account for de novo resistance to HER2 targeting therapies in basal-like BC carrying HER2 gene amplification.

Metformin: Targeting the Metabolo-Epigenetic Link in Cancer Biology.

Metabolism can directly drive or indirectly enable an aberrant chromatin state of cancer cells. The physiological and molecular principles of the metabolic link to epigenetics provide a basis for pharmacological modulation with the anti-diabetic biguanide metformin. Here, we briefly review how metabolite-derived chromatin modifications and the metabolo-epigenetic machinery itself are both amenable to modification by metformin in a local and a systemic manner. First, we consider the capacity of metformin to target global metabolic pathways or specific metabolic enzymes producing chromatin-modifying metabolites. Second, we examine its ability to directly or indirectly fine-tune the activation status of chromatin-modifying enzymes. Third, we envision how the interaction between metformin, diet and gut microbiota might systemically regulate the metabolic inputs to chromatin. Experimental and clinical validation of metformin's capacity to change the functional outcomes of the metabolo-epigenetic link could offer a proof-of-concept to therapeutically test the metabolic adjustability of the epigenomic landscape of cancer.

Silibinin and SARS-CoV-2: Dual Targeting of Host Cytokine Storm and Virus Replication Machinery for Clinical Management of COVID-19 Patients.

COVID-19, the illness caused by infection with the novel coronavirus SARS-CoV-2, is a rapidly spreading global pandemic in urgent need of effective treatments. Here we present a comprehensive examination of the host- and virus-targeted functions of the flavonolignan silibinin, a potential drug candidate against COVID-19/SARS-CoV-2. As a direct inhibitor of STAT3-a master checkpoint regulator of inflammatory cytokine signaling and immune response-silibinin might be expected to phenotypically integrate the mechanisms of action of IL-6-targeted monoclonal antibodies and pan-JAK1/2 inhibitors to limit the cytokine storm and T-cell lymphopenia in the clinical setting of severe COVID-19. As a computationally predicted, remdesivir-like inhibitor of RNA-dependent RNA polymerase (RdRp)-the central component of the replication/transcription machinery of SARS-CoV-2-silibinin is expected to reduce viral load and impede delayed interferon responses. The dual ability of silibinin to target both the host cytokine storm and the virus replication machinery provides a strong rationale for the clinical testing of silibinin against the COVID-19 global public health emergency. A randomized, open-label, phase II multicentric clinical trial (SIL-COVID19) will evaluate the therapeutic efficacy of silibinin in the prevention of acute respiratory distress syndrome in moderate-to-severe COVID-19-positive onco-hematological patients at the Catalan Institute of Oncology in Catalonia, Spain.

Tumor Cell-Intrinsic Immunometabolism and Precision Nutrition in Cancer Immunotherapy.

One of the greatest challenges in the cancer immunotherapy field is the need to biologically rationalize and broaden the clinical utility of immune checkpoint inhibitors (ICIs). The balance between metabolism and immune response has critical implications for overcoming the major weaknesses of ICIs, including their lack of universality and durability. The last decade has seen tremendous advances in understanding how the immune system's ability to kill tumor cells requires the conspicuous metabolic specialization of T-cells. We have learned that cancer cell-associated metabolic activities trigger shifts in the abundance of some metabolites with immunosuppressory roles in the tumor microenvironment. Yet very little is known about the tumor cell-intrinsic metabolic traits that control the immune checkpoint contexture in cancer cells. Likewise, we lack a comprehensive understanding of how systemic metabolic perturbations in response to dietary interventions can reprogram the immune checkpoint landscape of tumor cells. We here review state-of-the-art molecular- and functional-level interrogation approaches to uncover how cell-autonomous metabolic traits and diet-mediated changes in nutrient availability and utilization might delineate new cancer cell-intrinsic metabolic dependencies of tumor immunogenicity. We propose that clinical monitoring and in-depth molecular evaluation of the cancer cell-intrinsic metabolic traits involved in primary, adaptive, and acquired resistance to cancer immunotherapy can provide the basis for improvements in therapeutic responses to ICIs. Overall, these approaches might guide the use of metabolic therapeutics and dietary approaches as novel strategies to broaden the spectrum of cancer patients and indications that can be effectively treated with ICI-based cancer immunotherapy.

The LSD1 inhibitor iadademstat (ORY-1001) targets SOX2-driven breast cancer stem cells: a potential epigenetic therapy in luminal-B and HER2-positive breast cancer subtypes.

SOX2 is a core pluripotency-associated transcription factor causally related to cancer initiation, aggressiveness, and drug resistance by driving the self-renewal and seeding capacity of cancer stem cells (CSC). Here, we tested the ability of the clinically proven inhibitor of the lysine-specific demethylase 1 (LSD1/KDM1A) iadademstat (ORY-100) to target SOX2-driven CSC in breast cancer. Iadademstat blocked CSC-driven mammosphere formation in breast cancer cell lines that are dependent on SOX2 expression to maintain their CSC phenotype. Iadademstat prevented the activation of an LSD1-targeted stemness-specific SOX2 enhancer in CSC-enriched 3-dimensional spheroids. Using high-throughput transcriptional data available from the METABRIC dataset, high expression of SOX2 was significantly more common in luminal-B and HER2-enriched subtypes according to PAM50 classifier and in IntClust1 (high proliferating luminal-B) and IntClust 5 (luminal-B and HER2-amplified) according to integrative clustering. Iadademstat significantly reduced mammospheres formation by CSC-like cells from a multidrug-resistant luminal-B breast cancer patient-derived xenograft but not of those from a treatment-naïve luminal-A patient. Iadademstat reduced the expression of SOX2 in luminal-B but not in luminal-A mammospheres, likely indicating a selective targeting of SOX2-driven CSC. The therapeutic relevance of targeting SOX2-driven breast CSC suggests the potential clinical use of iadademstat as an epigenetic therapy in luminal-B and HER2-positive subtypes.

Resveratrol targets PD-L1 glycosylation and dimerization to enhance antitumor T-cell immunity.

New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.