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Figure S1. PARD3-inactivation in the H157 lung cancer cell line. Figure S2. Ratio charts of the multiplex ligation-dependent probe amplification (MLPA) depicting a large intragenic deletion (from exon 4 to 23) in one of the LSCCs but not a normal DNA. Figure S3. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to determine the presence of promoter hypermethylation of PARD3. Figure S4. Relative abundance of the PARD3 transcriptsFigure S5. Western blot showing ectopic and transient expression of the indicated PAR3 proteins in the H157 cell line, immunoblotted with HA or PAR3 antibodies. Figure S6. Immunofluorescence with the anti-PAR3 antibody in the indicated T98G-derived cells, after (Dox+) induction of PAR3 expression with doxycycline (1 ng/µl, 24h). Scale bar, 50 µm. Figure S7. Western blot depicts the endogenous PAR3 protein in the indicated lung cancer cells and the ectopic expression of PAR3 in the H157tr-pD41_R74del cells (dox, 1 ng/µl; 24 h). Figure S8. Immunostaining of PAR3 in lung primary tumors and head and neck squamous cell carcinomas (HNSCCs).

Correct apicobasal polarization and intercellular adhesions are essential for the appropriate development of normal epithelia. Here, we investigated the contribution of the cell polarity regulator PARD3 to the development of lung squamous cell carcinomas (LSCC). Tumor-specific PARD3 alterations were found in 8% of LSCCs examined, placing PARD3 among the most common tumor suppressor genes in this malignancy. Most PAR3-mutant proteins exhibited a relative reduction in the ability to mediate formation of tight junctions and actin-based protrusions, bind atypical protein kinase C, activate RAC1, and activate STAT3 at cell confluence. Thus, PARD3 alterations prevented the formation of contacts between neighboring cells and the subsequent downstream signaling. Notably, reconstituting PAR3 activity in vivo reduced tumor-invasive and metastatic properties. Our findings define PARD3 as a recurrently inactivated cell polarity regulator in LSCC that affects tumor aggressiveness and metastasis. Cancer Res; 75(7); 1287–97. ©2015 AACR.

Supplementary Materials and Methods

Table S1. List of lung cancer cell lines and histopathological type used to determine the status of the PARD3. Table S2. List of primers used for cloning, PCR and Sanger sequencing of PARD3. Table S3. List of single nucleotide polymorphisms (SNPs) and variants of unknown biological significance identified in lung cancer cell lines and lung primary tumors tested. Table S4. Clinical and pathological parameters of the NSCLC patients and tumors included in the PARD3 gene alterations screening. Table S5. List of genes selected from those genes that fulfilled the criteria: i) adjusted p

CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvβ3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6β1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvβ3 or α6β1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvβ3 and α6β1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvβ3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6β1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiolindependent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6β1-bindingdefective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6β1, but not via αvβ3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate itstranscriptional activity in ER+ BC cells

Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-RasV12-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial–cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. “Upper” and “lower” glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl–carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK–TKI therapy-induced EMT promotes cross-resistance to new-generation ALK–TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK–TKI crizotinib exhibited increased resistance to secondgeneration brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK–TKIs, which was partially recapitulated upon chronic TGF stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGF )/SMAD signaling pathway. Silibinin deactivated TGF -regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGF type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGF into the extracellular fluid of ALK–TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK–TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK–TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGF /SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells., Ministry of Science and Innovation, Spain (MICINN) Spanish Government, Plan Nacional de l+D+I PID2019-10455GB-I00 CP20/00003 Spanish Government, Fundacio Oncolliga Girona (Lliga catalana d'ajuda al malalt de cancer, Girona), Spanish Government, Center for Forestry Research & Experimentation (CIEF), European Commission PI22/00297, Grupo Espanol de Cancer de Pulmon (GECP) RTI2019-096724-B-C21, La Marato de TV3 foundation, Health Research and Innovation Strategic Plan PROMETEO/2021/059, Pla strategic de recerca i innovacio en salut 201906, Generalitat de Catalunya, Instituto de Salud Carlos III SLT006/17/114

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.

Progesterone receptor (PR) isoforms can drive unique phenotypes in luminal breast cancer (BC). Here, we hypothesized that PR-B and PR-A isoforms differentially modify the cross-talk between prolactin and fatty acid synthase (FASN) in BC. We profiled the responsiveness of the FASN gene promoter to prolactin in T47Dco BC cells constitutively expressing PR-A and PR-B, in the PR-null variant T47D-Y cell line, and in PR-null T47D-Y cells engineered to stably re-express PR-A (T47D-YA) or PR-B (T47D-YB). The capacity of prolactin to up-regulate FASN gene promoter activity in T47Dco cells was lost in T47D-Y and TD47-YA cells. Constitutively up-regulated FASN gene expression in T47-YB cells and its further stimulation by prolactin were both suppressed by the prolactin receptor antagonist hPRL-G129R. The ability of the FASN inhibitor C75 to decrease prolactin secretion was more conspicuous in T47-YB cells. In T47D-Y cells, which secreted notably less prolactin and downregulated prolactin receptor expression relative to T47Dco cells, FASN blockade resulted in an augmented secretion of prolactin and up-regulation of prolactin receptor expression. Our data reveal unforeseen PR-B isoform-specific regulatory actions in the cross-talk between prolactin and FASN signaling in BC. These findings might provide new PR-B/FASN-centered predictive and therapeutic modalities in luminal intrinsic BC subtypes Fil: Menendez, Javier A.. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Peirce, Susan K.. Clemson University. Pearce Center Professional Communication; Estados Unidos Fil: Papadimitropoulou, Adriana. Biomedical Research Foundation Academy of Athens; Grecia Fil: Cuyàs, Elisabet. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Steen, Travis Vander. Mayo Foundation for Medical Education and Research. Mayo Clinic; Estados Unidos Fil: Verdura, Sara. Institut Català d'Oncologia; España. Institut d’Investigació Biomèdica de Girona; España Fil: Vellón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Chen, Wen Y.. Clemson University; Estados Unidos Fil: Lupu, Ruth. Mayo Foundation for Medical Education and Research. Mayo Clinic; Estados Unidos. Clemson University; Estados Unidos. Mayo Clinic Cancer Center; Estados Unidos

Altres ajuts: Fundació Oncolliga Girona, Generalitat Valenciana (PROMETEO/2016/006) 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.

Simple Summary Nintedanib is an anti-angiogenic agent that has received approval in the European Union for the treatment of non-small cell lung cancer (NSCLC) after first-line chemotherapy. Here, we explore the possibility that the flavonolignan silibinin—the major bioactive component from the seeds of the milk thistle herb (Silybum marianum)—can provide clinical benefit in patients with advanced NSCLC treated with nintedanib. In vitro studies revealed that silibinin targets biological functions important for the therapeutic efficacy of nintedanib; specifically, activation of the transcription factor STAT3 and sequestration into lysosomal “safe houses”. Supplementation with the silibinin-based nutraceutical Legasil® to patients with NSCLC receiving nintedanib/docetaxel was associated with increased clinical responses and a significantly longer time to treatment failure. Our findings provide a biological and clinical rationale for combining silibinin with nintedanib in a patient population for whom few effective second-line chemotherapy regimens are available. Abstract The anti-angiogenic agent nintedanib has been shown to prolong overall and progression-free survival in patients with advanced non-small-cell lung cancer (NSCLC) who progress after first-line platinum-based chemotherapy and second-line immunotherapy. Here, we explored the molecular basis and the clinical benefit of incorporating the STAT3 inhibitor silibinin—a flavonolignan extracted from milk thistle—into nintedanib-based schedules in advanced NSCLC. First, we assessed the nature of the tumoricidal interaction between nintedanib and silibinin and the underlying relevance of STAT3 activation in a panel of human NSCLC cell lines. NSCLC cells with poorer cytotoxic responses to nintedanib exhibited a persistent, nintedanib-unresponsive activated STAT3 state, and deactivation by co-treatment with silibinin promoted synergistic cytotoxicity. Second, we tested whether silibinin could impact the lysosomal sequestration of nintedanib, a lung cancer cell-intrinsic mechanism of nintedanib resistance. Silibinin partially, but significantly, reduced the massive lysosomal entrapment of nintedanib occurring in nintedanib-refractory NSCLC cells, augmenting the ability of nintedanib to reach its intracellular targets. Third, we conducted a retrospective, observational multicenter study to determine the efficacy of incorporating an oral nutraceutical product containing silibinin in patients with NSCLC receiving a nintedanib/docetaxel combination in second- and further-line settings (n = 59). Overall response rate, defined as the combined rates of complete and partial responses, was significantly higher in the study cohort receiving silibinin supplementation (55%) than in the control cohort (22%, p = 0.011). Silibinin therapy was associated with a significantly longer time to treatment failure in multivariate analysis (hazard ratio 0.43, p = 0.013), despite the lack of overall survival benefit (hazard ratio 0.63, p = 0.190). Molecular mechanisms dictating the cancer cell-intrinsic responsiveness to nintedanib, such as STAT3 activation and lysosomal trapping, are amenable to pharmacological intervention with silibinin. A prospective, powered clinical trial is warranted to confirm the clinical relevance of these findings in patients with advanced NSCLC.

Catechol-containing polyphenols present in coffee and tea, while serving as excellent substrates for catechol-O-methyltransferase (COMT)-catalyzed O-methylation, can also operate as COMT inhibitors. However, little is known about the relationship between COMT and the characteristic phenolics present in extra virgin olive oil (EVOO). We here selected the EVOO dihydroxy-phenol oleacein for a computational study of COMT-driven methylation using classic molecular docking/molecular dynamics simulations and hybrid quantum mechanical/molecular mechanics, which were supported by in vitro activity studies using human COMT. Oleacein could be superimposed onto the catechol-binding site of COMT, maintaining the interactions with the atomic positions involved in methyl transfer from the S-adenosyl-L-methionine cofactor. The transition state structure for the meta-methylation in the O5 position of the oleacein benzenediol moiety was predicted to occur preferentially. Enzyme analysis of the conversion ratio of catechol to O-alkylated guaiacol confirmed the inhibitory effect of oleacein on human COMT, which remained unaltered when tested against the protein version encoded by the functional Val158Met polymorphism of the COMT gene. Our study provides a theoretical determination of how EVOO dihydroxy-phenols can be metabolized via COMT. The ability of oleacein to inhibit COMT adds a new dimension to the physiological and therapeutic utility of EVOO secoiridoids.

The METTEN study assessed the efficacy, tolerability, and safety of adding metformin to neoadjuvant chemotherapy plus trastuzumab in early HER2-positive breast cancer (BC). Women with primary, non-metastatic HER2-positive BC were randomized (1:1) to receive metformin (850 mg twice-daily) for 24 weeks concurrently with 12 cycles of weekly paclitaxel plus trastuzumab, followed by four cycles of 3-weekly FE75C plus trastuzumab (arm A), or equivalent regimen without metformin (arm B), followed by surgery. Primary endpoint was the rate of pathological complete response (pCR) in the per-protocol efficacy population. pCR rate was numerically higher in the metformin-containing arm A (19 of 29 patients [65.5%, 95% CI: 47.3–80.1]) than in arm B (17 of 29 patients [58.6%, 95% CI: 40.7–74.5]; OR 1.34 [95% CI: 0.46–3.89], P = 0.589). The rate of breast-conserving surgery was 79.3% and 58.6% in arm A and B (P = 0.089), respectively. Blood metformin concentrations (6.2 μmol/L, 95% CI: 3.6–8.8) were within the therapeutic range. Seventy-six percent of patients completed the metformin-containing regimen; 13% of patients in arm A dropped out because of metformin-related gastrointestinal symptoms. The most common adverse events (AEs) of grade ≥3 were neutropenia in both arms and diarrhea in arm A. None of the serious AEs was deemed to be metformin-related. Addition of anti-diabetic doses of metformin to a complex neoadjuvant regimen was well tolerated and safe. Because the study was underpowered relative to its primary endpoint, the efficacy data should be interpreted with caution.

Triple-negative/basal-like breast cancer (BC) is characterized by aggressive biological features, which allow relapse and metastatic spread to occur more frequently than in hormone receptor-positive (luminal) subtypes. The molecular complexity of triple-negative/basal-like BC poses major challenges for the implementation of targeted therapies, and chemotherapy remains the standard approach at all stages. The matricellular protein cysteine-rich angiogenic inducer 61 (CCN1/CYR61) is associated with aggressive metastatic phenotypes and poor prognosis in BC, but it is unclear whether anti-CCN1 approaches can be successfully applied in triple-negative/basal-like BC. Herein, we first characterized the prevalence of CNN1 expression in matched samples of primary tumors and metastatic relapse in a series of patients with BC. We then investigated the biological effect of CCN1 depletion on tumorigenic traits in vitro and in vivo using archetypal TNBC cell lines. Immunohistochemical analyses of tissue microarrays revealed a significant increase of the highest CCN1 score in recurrent tissues of triple-negative/basal-like BC tumors. Stable silencing of CCN1 in triple-negative/basal-like BC cells promoted a marked reduction in the expression of the CCN1 integrin receptor α(v)β(3), inhibited anchorage-dependent cell growth, reduced clonogenicity, and impaired migration capacity. In an orthotopic model of triple-negative/basal-like BC, silencing of CCN1 notably reduced tumor burden, which was accompanied by decreased microvessel density and concurrent induction of the luminal epithelial marker E-cadherin. Thus, CNN1/CYR61-targeting strategies might have therapeutic value in suppressing the biological aggressiveness of triple-negative/basal-like BC.

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting., The authors would like to thank Dr. Kenneth McCreath for editorial support. This work was supported by the NIH National Cancer Institute Grants R01 CA116623 (to Ruth Lupu) and R01 CA166741 (to Scott H. Kaufmann) and by the U.S. Department of Defense (DOD)-Breakthrough 3 Grants BC151072 and BC151072P1 (to Ruth Lupu). Work in the Menendez laboratory is supported by the Spanish Ministry of Science and Innovation (Grants SAF2016-80639-P and PID2019-10455GB-I00, Plan Nacional de l + D + I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona). Joan Montero acknowledges support from the Ramon y Cajal Programme, Ministerio de Economía y Competitividad (RYC-2015-18357) and the Spanish National Plan “Retos Investigación” I + D + I (RTI2018-094533-A-I00) from the Ministerio de Ciencia, Innovación y Universidades. Elisabet Cuyàs holds a research contract “Miguel Servet” (CP20/00003) from the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (Spain). All authors have read and agreed to the published version of the manuscript.

Simple Summary Overactivation of the human epidermal growth factor receptor 2 (HER2) is one of the main drivers of tamoxifen resistance in estrogen receptor (ER)-positive breast cancer patients. Combined targeting of HER2 and ER, however, has yielded disappointing results in the clinical setting. Therefore, other potential mechanisms for tamoxifen resistance would not be overcome by solely blocking the cross-talk between ER and HER2 at the receptor(s) level. Using cell lines, animal models, and clinical data, we provide evidence to support a critical role of fatty acid synthase (FASN)—the major site for endogenous fat synthesis—in HER2-driven tamoxifen resistance. Importantly, treatment with a FASN inhibitor impeded the estrogen-like tumor-promoting effects of tamoxifen and fully restored the anti-estrogenic activity of tamoxifen in ER+/HER2-overexpressing breast cancer xenografts. We postulate FASN as a biological determinant of HER2-driven tamoxifen resistance and FASN inhibition as a novel therapeutic approach to restore tamoxifen sensitivity in endocrine-resistant breast cancer. Abstract The identification of clinically important molecular mechanisms driving endocrine resistance is a priority in estrogen receptor-positive (ER+) breast cancer. Although both genomic and non-genomic cross-talk between the ER and growth factor receptors such as human epidermal growth factor receptor 2 (HER2) has frequently been associated with both experimental and clinical endocrine therapy resistance, combined targeting of ER and HER2 has failed to improve overall survival in endocrine non-responsive disease. Herein, we questioned the role of fatty acid synthase (FASN), a lipogenic enzyme linked to HER2-driven breast cancer aggressiveness, in the development and maintenance of hormone-independent growth and resistance to anti-estrogens in ER/HER2-positive (ER+/HER2+) breast cancer. The stimulatory effects of estradiol on FASN gene promoter activity and protein expression were blunted by anti-estrogens in endocrine-responsive breast cancer cells. Conversely, an AKT/MAPK-related constitutive hyperactivation of FASN gene promoter activity was unaltered in response to estradiol in non-endocrine responsive ER+/HER2+ breast cancer cells, and could be further enhanced by tamoxifen. Pharmacological blockade with structurally and mechanistically unrelated FASN inhibitors fully impeded the strong stimulatory activity of tamoxifen on the soft-agar colony forming capacity—an in vitro metric of tumorigenicity—of ER+/HER2+ breast cancer cells. In vivo treatment with a FASN inhibitor completely prevented the agonistic tumor-promoting activity of tamoxifen and fully restored its estrogen antagonist properties against ER/HER2-positive xenograft tumors in mice. Functional cancer proteomic data from The Cancer Proteome Atlas (TCPA) revealed that the ER+/HER2+ subtype was the highest FASN protein expressor compared to basal-like, HER2-enriched, and ER+/HER2-negative breast cancer groups. FASN is a biological determinant of HER2-driven endocrine resistance in ER+ breast cancer. Next-generation, clinical-grade FASN inhibitors may be therapeutically relevant to countering resistance to tamoxifen in FASN-overexpressing ER+/HER2+ breast carcinomas.

SummaryDespite the genetic inactivation of SMARCA4, a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. This is associated with impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, which confer a strong dependency on the KDM6s in the SMARCA4-mutant cells, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4-mutant lung cancer cells or primary small cell carcinoma of the hypercalcaemic type (SCCOHT) had strong anti-tumour effects. Our results highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4-mutant cancer patients.

HER2 transactivation by the HER3 ligand heregulin (HRG) promotes an endocrine-resistant phenotype in the estrogen receptor-positive (ER+) luminal-B subtype of breast cancer. The underlying biological mechanisms that link them are, however, incompletely understood. Here, we evaluated the putative role of the lipogenic enzyme fatty acid synthase (FASN) as a major cause of HRG-driven endocrine resistance in ER+/HER2-negative breast cancer cells. MCF-7 cells engineered to stably overexpress HRG (MCF-7/HRG), an in vitro model of tamoxifen/fulvestrant-resistant luminal B-like breast cancer, showed a pronounced up-regulation of FASN gene/FASN protein expression. Autocrine HRG up-regulated FASN expression via HER2 transactivation and downstream activation of PI-3K/AKT and MAPK-ERK1/2 signaling pathways. The HRG-driven FASN-overexpressing phenotype was fully prevented in MCF-7 cells expressing a structural deletion mutant of HRG that is sequestered in a cellular compartment and lacks the ability to promote endocrine-resistance in an autocrine manner. Pharmacological inhibition of FASN activity blocked the estradiol-independent and tamoxifen/fulvestrant-refractory ability of MCF-7/HRG cells to anchorage-independently grow in soft-agar. In vivo treatment with a FASN inhibitor restored the anti-tumor activity of tamoxifen and fulvestrant against fast-growing, hormone-resistant MCF-7/HRG xenograft tumors in mice. Overall, these findings implicate FASN as a key enabler for endocrine resistance in HRG+/HER2-breast cancer and highlight the therapeutic potential of FASN inhibitors for the treatment of endocrine therapy-resistant luminal-B breast cancer. Fil: Menendez, Javier A.. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España. Institut Català d'Oncologia ; España Fil: Mehmi, Inderjit. The Angeles Clinic And Research Institute; Estados Unidos Fil: Papadimitropoulou, Adriana. Academy of Athens; Grecia Fil: Steen, Travis Vander. Mayo Clinic; Estados Unidos Fil: Cuyàs, Elisabet. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España. Institut Català d'Oncologia ; España Fil: Verdura, Sara. Institut Català d'Oncologia ; España. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España Fil: Espinoza, Ingrid. University of Mississippi; Estados Unidos Fil: Vellón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Atlas, Ella. University of Ottawa; Canadá Fil: Lupu, Ruth. Mayo Clinic Cancer Center; Estados Unidos. Mayo Clinic; Estados Unidos. Academy of Athens; Grecia

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 (alpha-glucosidase/alpha-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.

Sustained HER2/HER3 signaling due to the overproduction of the HER3 ligand heregulin (HRG) is proposed as a key contributor to endocrine resistance in estrogen receptor-positive (ER+) breast cancer. The molecular mechanisms linking HER2 transactivation by HRG-bound HER3 to the acquisition of a hormone-independent phenotype in ER+ breast cancer is, however, largely unknown. Here, we explored the possibility that autocrine HRG signaling drives cytokine-related endocrine resistance in ER+ breast cancer cells. We used human cytokine antibody arrays to semi-quantitatively measure the expression level of 60 cytokines and growth factors in the extracellular milieu of MCF-7 cells engineered to overexpress full-length HRGβ2 (MCF-7/HRG cells). Interleukin-8 (IL-8), a chemokine closely linked to ER inaction, emerged as one the most differentially expressed cytokines. Cytokine profiling using structural deletion mutants lacking both the N-terminus and the cytoplasmic-transmembrane region of HRGβ2—which is not secreted and cannot transactivate HER2—or lacking a nuclear localization signal at the N-terminus—which cannot localize at the nucleus but is actively secreted and transactivates HER2—revealed that the HRG-driven activation of IL-8 expression in ER+ cells required HRG secretion and transactivation of HER2 but not HRG nuclear localization. The functional blockade of IL-8 with a specific antibody inversely regulated ERα-driven transcriptional activation in endocrine-sensitive MCF-7 cells and endocrine-resistant MCF-7/HRG cells. Overall, these findings suggest that IL-8 participates in the HRG-driven endocrine resistance program in ER+/HER2- breast cancer and might illuminate a potential clinical setting for IL8- or CXCR1/2-neutralizing antibodies. Fil: Papadimitropoulou, Adriana. Academy of Athens; Grecia Fil: Vellón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Atlas, Ella. University of Ottawa; Canadá Fil: Steen, Travis Vander. Mayo Clinic; Estados Unidos Fil: Cuyàs, Elisabet. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España. Institut Català d'Oncologia; España Fil: Verdura, Sara. Institut Català d'Oncologia; España. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España Fil: Espinoza, Ingrid. University of Mississippi; Estados Unidos Fil: Menendez, Javier A.. Institut Català d'Oncologia; España. Institut D'investigació Biomèdica de Girona Dr. Josep Trueta; España Fil: Lupu, Ruth. Mayo Clinic; Estados Unidos. University of Ottawa; Canadá. Mayo Clinic Cancer Center; Estados Unidos

Altres ajuts: "La Marató TV3" 201906 - Health Research and Innovation Strategic Plan (SLT006/17/114; PERIS 2016-2020; Pla stratègic de recerca i innovació en salut; Departament de Salut, Generalitat de Catalunya). 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.

Unraveling the key mechanisms governing the retention versus loss of the cancer stem cell (CSC) state would open new therapeutic avenues to eradicate cancer. Mitochondria are increasingly recognized key drivers in the origin and development of CSC functional traits. We here propose the new term “mitostemness” to designate the mitochondria-dependent signaling functions that, evolutionary rooted in the bacterial origin of mitochondria, regulate the maintenance of CSC self-renewal and resistance to differentiation. Mitostemness traits, namely mitonuclear communication, mitoproteome components, and mitochondrial fission/fusion dynamics, can be therapeutically exploited to target the CSC state. We briefly review the pre-clinical evidence of action of investigational compounds on mitostemness traits and discuss ongoing strategies to accelerate the clinical translation of new mitostemness drugs. The recognition that the bacterial origin of present-day mitochondria can drive decision-making signaling phenomena may open up a new therapeutic dimension against life-threatening CSCs. New therapeutics aimed to target mitochondria not only as biochemical but also as biophysical and morpho-physiological hallmarks of CSC might certainly guide improvements to cancer treatment.

Background: Epidemiological, pre-clinical, and window-of-opportunity trials have suggested that the anti-diabetic drug metformin (MET) may have beneficial effects on breast cancer (BC). The clinical relevance of combining MET with current standards of care for first-line treatment of BC is unknown. We investigated the safety and efficacy of adding MET to neoadjuvant chemotherapy plus trastuzumab (TTZ) in patients with HER2-positive early BC. Methods: In a randomized, multicenter, open-label phase 2 study, women with operable, locally advanced, or inflammatory HER2+ BC were randomly assigned in a 1:1 ratio to receive daily MET (1,700 mg) for 24 weeks concurrently with 12 cycles of weekly paclitaxel plus TTZ followed by four cycles of fluorouracil, epirubicin, cyclophosphamide/3w plus TTZ (arm A) or equivalent sequential chemotherapy plus TTZ without MET (arm B), followed by surgery. Patients were stratified by age, extent of disease (cT2 cN0-1 vs ≥ T3 or ≥ N2), and hormone receptor (HR)-status. The intention-to-treat (ITT) safety population comprised all women who underwent randomization and received at least one treatment dose. Primary endpoint was the rate of pathological complete response (pCR) analyzed in the per-protocol (PP) efficacy population. pCR was defined as absence of invasive cancer in the breast and axillary nodes, irrespective of carcinoma in situ (ypT0/is ypN0). Results: From June 1, 2012 to March 17, 2016, 98 patients were assessed for eligibility at 10 centers in Spain. Of 84 (85.7%) patients who were randomized, 41 patients were allocated to arm A and 43 to arm B. The most common adverse effects (AEs) in the 79 ITT patients were fatigue, diarrhea, nausea, alopecia, sensory neuropathy, mucositis, neutropenia, and elevated AST/ALT. Most AEs were grades 1–2 (>90% in both arms). The most common AEs grade ≥3 were neutropenia (7/38 women in arm A and 5/41 women in arm B) and diarrhea (5 and 0, respectively). The number of serious AEs was 3 in arm A (none of them was deemed to be MET-related) and 2 in arm B. At week 12, one (2.9%) patient in arm A and 6 (15%) in arm B exhibited asymptomatic decreases in the left ventricular ejection fraction (LVEF). At week 24, none (0%) of the patients in arm A and 3 (7.9%) patients in arm B presented decreases of LVEF below 50% and >10% from baseline. Only one patient (2.7%) in arm B experienced symptomatic heart failure. The rates of breast-conserving surgery were 79.3% and 58.6% in PP arms A and B, respectively. Also, 19/29 PP patients in arm A (65.5%, 95% CI 47–80) had a pCR versus 17/29 PP patients (58.6%, 95% CI 41–74) in arm B (OR 1.34 [95% CI 0.46–3.89], p=0.589). The combined rates of pCR and near-pCR (ypT1aN0) were 79.3% in arm A and 72.4% in arm B. Significantly fewer pCR were noted for tumors that were HR-positive regardless of which arm the patients were randomized. Conclusion: The higher pCR rate in HER2+ BC patients receiving MET-containing neoadjuvant therapy and TTZ did not reach statistical significance. Evaluation of long-term outcome data such as 5-year disease-free survival and correlative biological studies are needed to evaluate the clinico-molecular relevance of these findings. Citation Format: Pernas S, Dorca J, Álvarez-López I, Martínez S, Saura C, Batista López N, Rodríguez-Sánchez CA, Amillano K, Domínguez-Fernández S, Luque Cabal M, Morilla I, Viñas G, Cortés J, Cuyàs E, Verdura S, Fernández-Arroyo S, Joven J, Pérez E, García M, Bosch N, López-Bonet E, Saidani S, Buxó M, Menendez JA, Martin-Castillo B. Safety and efficacy of neoadjuvant metformin with trastuzumab and chemotherapy in women with HER2-positive early breast cancer: A randomized, open-label, multicenter, phase 2 trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-01.

The flavonolignan silibinin, the major bioactive component of the silymarin extract of Silybum marianum (milk thistle) seeds, is gaining traction as a novel anti-cancer therapeutic. Here, we review the historical developments that have laid the groundwork for the evaluation of silibinin as a chemopreventive and therapeutic agent in human lung cancer, including translational insights into its mechanism of action to control the aggressive behavior of lung carcinoma subtypes prone to metastasis. First, we summarize the evidence from chemically induced primary lung tumors supporting a role for silibinin in lung cancer prevention. Second, we reassess the preclinical and clinical evidence on the effectiveness of silibinin against drug resistance and brain metastasis traits of lung carcinomas. Third, we revisit the transcription factor STAT3 as a central tumor-cell intrinsic and microenvironmental target of silibinin in primary lung tumors and brain metastasis. Finally, by unraveling the selective vulnerability of silibinin-treated tumor cells to drugs using CRISPR-based chemosensitivity screenings (e.g., the hexosamine biosynthesis pathway inhibitor azaserine), we illustrate how the therapeutic use of silibinin against targetable weaknesses might be capitalized in specific lung cancer subtypes (e.g., KRAS/STK11 co-mutant tumors). Forthcoming studies should take up the challenge of developing silibinin and/or next-generation silibinin derivatives as novel lung cancer-preventive and therapeutic biomolecules.

International audience; The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

// Elisabet Cuyas 1,2,* , Sara Verdura 1,2,* , Salvador Fernandez-Arroyo 3,4 , Joaquim Bosch-Barrera 5 , Begona Martin-Castillo 6 , Jorge Joven 3,4 and Javier A. Menendez 1,2 1 Metabolism and Cancer Group, Program Against Cancer Therapeutic Resistance, Catalan Institute of Oncology, Girona, Spain 2 Molecular Oncology Group, Girona Biomedical Research Institute (IDIBGI), Girona, Spain 3 Unitat de Recerca Biomedica, Hospital Universitari de Sant Joan, Institut d’Investigacio Sanitaria Pere Virgili, Universitat Rovira i Virgili, Reus, Catalonia, Spain 4 Campus of International Excellence Southern Catalonia, Tarragona, Catalonia, Spain 5 Medical Oncology, Catalan Institute of Oncology, Girona, Catalonia, Spain 6 Unit of Clinical Research, Catalan Institute of Oncology, Girona, Spain * These authors have contributed equally to this work Correspondence to: Javier A. Menendez, email: // Keywords : cancer stem cells; metabolic maps; tumor heterogeneity Received : July 21, 2017 Accepted : September 29, 2017 Published : October 15, 2017 Abstract Personalized cancer medicine based on the analysis of tumors en masse is limited by tumor heterogeneity, which has become a major obstacle to effective cancer treatment. Cancer stem cells (CSC) are emerging as key drivers of inter- and intratumoral heterogeneity. CSC have unique metabolic dependencies that are required not only for specific bioenergetic/biosynthetic demands but also for sustaining their operational epigenetic traits, i.e. self-renewal, tumor-initiation, and plasticity. Given that the metabolome is the final downstream product of all the –omic layers and, therefore, most representative of the biological phenotype, we here propose that a novel approach to better understand the complexity of tumor heterogeneity is by mapping and cataloging small numbers of CSC metabolomic phenotypes. The narrower metabolomic diversity of CSC states could be employed to reduce multidimensional tumor heterogeneity into dynamic models of fewer actionable sub-phenotypes. The identification of the driver nodes that are used differentially by CSC states to metabolically regulate self-renewal and tumor initation and escape chemotherapy might open new preventive and therapeutic avenues. The mapping of CSC metabolomic states could become a pioneering strategy to reduce the dimensionality of tumor heterogeneity and improve our ability to examine changes in tumor cell populations for cancer detection, prognosis, prediction/monitoring of therapy response, and detection of therapy resistance and recurrent disease. The identification of driver metabolites and metabolic nodes accounting for a large amount of variance within the CSC metabolomic sub-phenotypes might offer new unforeseen opportunities for reducing and exploiting tumor heterogeneity via metabolic targeting of CSC.

The development of a single immuno-metabolic adjuvant capable of modulating, in the appropriate direction and intensity, the complex antagonistic and symbiotic interplays between tumor cells, immune cells, and the gut microbiota may appear pharmacologically implausible. Metformin might help solve this conundrum and beneficially impact the state of cancer-immune system interactions Work in the Menendez laboratory is supported by the Spanish Ministry of Science and Innovation (Grant SAF2016-80639-P, Plan Nacional de l +D+I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona)

The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 umol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors., Work in the Menendez laboratory is supported by the Spanish Ministry of Science and Innovation (Grant SAF2016-80639-P, Plan Nacional de l+D+I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona). The Spanish Ministry of Economy and Competitiveness (MINECO, Project RTI2018-096724-B-C21) and the Generalitat Valenciana (PROMETEO/2016/006) supports work in the Encinar laboratory

Homocisteïna; Càncer de mama; Dieta cetogènica Homocisteína; Cáncer de mama; Dieta cetogénica Homocysteine; Breast cancer; Ketogenic diet Certain dietary interventions might improve the therapeutic index of cancer treatments. An alternative to the “drug plus diet” approach is the pharmacological reproduction of the metabolic traits of such diets. Here we explored the impact of adding metformin to an established therapeutic regimen on the systemic host metabolism of cancer patients. A panel of 11 serum metabolites including markers of mitochondrial function and intermediates/products of folate-dependent one-carbon metabolism were measured 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. Metabolite profiles revealed a significant increase of the ketone body β-hydroxybutyrate and of the TCA intermediate α-ketoglutarate in the metformin-containing arm. A significant relationship was found between the follow-up levels of homocysteine and the ability of treatment arms to achieve a pathological complete response (pCR). In the metformin-containing arm, patients with significant elevations of homocysteine tended to have a higher probability of pCR. The addition of metformin to an established anti-cancer therapeutic regimen causes a fasting-mimicking modification of systemic host metabolism. Circulating homocysteine could be explored as a clinical pharmacodynamic biomarker linking the antifolate-like activity of metformin and biological tumor response. This work was supported by grants from the Ministerio de Sanidad, Servicios Sociales e Igualdad (EC10-125, Ayudas para el Fomento de la Investigación Clínica Independiente to Begoña Martin-Castillo). Work in the Menendez laboratory is supported by the Ministerio de Ciencia e Innovación (Grant SAF2016-80639-P, Plan Nacional de l+D+I, founded by the European Regional Development Fund [EU FEDER], Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona).

An ever-growing number of preclinical studies have investigated the tumoricidal activity of the milk thistle flavonolignan silibinin. The clinical value of silibinin as a bona fide anti-cancer therapy, however, remains uncertain with respect to its bioavailability and blood–brain barrier (BBB) permeability. To shed some light on the absorption and bioavailability of silibinin, we utilized the Caco-2 cell monolayer model of human intestinal absorption to evaluate the permeation properties of three different formulations of silibinin: silibinin-meglumine, a water-soluble form of silibinin complexed with the amino-sugar meglumine; silibinin-phosphatidylcholine, the phytolipid delivery system Siliphos; and Eurosil85/Euromed, a milk thistle extract that is the active component of the nutraceutical Legasil with enhanced bioavailability. Our approach predicted differential mechanisms of transport and blood–brain barrier permeabilities between the silibinin formulations tested. Our assessment might provide valuable information about an idoneous silibinin formulation capable of reaching target cancer tissues and accounting for the observed clinical effects of silibinin, including a recently reported meaningful central nervous system activity against brain metastases Work in the Menendez laboratory was supported by the Spanish Ministry of Science and Innovation (Grant SAF2016-80639-P, Plan Nacional de l+D+I, founded by the European Regional Development Fund, Spain) and by an unrestricted research grant from the Fundació Oncolliga Girona (Lliga catalana d’ajuda al malalt de càncer, Girona). Work in the Vicente Micol laboratory was supported by Grants AGL2015-67995-C3-1-R from the Spanish Ministry of Economy and Competitiveness (MINECO); PROMETEO/2016/006, ACOMP/2013/093, ACIF/2013/064, ACIF/2015/158, APOTIP/2017/003, and APOSTD/2018/097 (Generalitat Valenciana), and CIBER (CB12/03/30038, Fisiopatologia de la Obesidad y la Nutricion, CIBERobn, Instituto de Salud Carlos III. Spain). Joaquim Bosch-Barrera is the recipient of a Grant from the Health Research and Innovation Strategic Plan (SLT006/17/114; PERIS 2016-2020; Pla stratègic de recerca i innovació en salut; Departament de Salut, Generalitat de Catalunya).

Altres ajuts: CERCA Programme/Generalitat de Catalunya Altres ajuts: Fundació Oncolliga Girona (Lliga catalana d'ajuda al malalt de càncer, Girona) Altres ajuts: Obra Social La Caixa Foundation on Collaborative Mathematics awarded to the Centre de Recerca Matemàtica (CRM) Therapeutic strategies targeting the hallmarks of aging can be broadly grouped into four categories, namely systemic (blood) factors, metabolic manipulation (diet regimens and dietary restriction mimetics), suppression of cellular senescence (senolytics), and cellular reprogramming, which likely have common characteristics and mechanisms of action. In evaluating the potential synergism of combining such strategies, however, we should consider the possibility of constraining trade-off phenotypes such as impairment in wound healing and immune response, tissue dysfunction and tumorigenesis. Moreover, we are rapidly learning that the benefit/risk ratio of aging-targeted interventions largely depends on intra- and inter-individual variations of susceptibility to the healthspan-, resilience-, and/or lifespan-promoting effects of the interventions. Here, we exemplify how computationally-generated proxies of the efficacy of a given lifespan/healthspan-promoting approach can predict the impact of baseline epigenetic heterogeneity on the positive outcomes of ketogenic diet and mTOR inhibition as single or combined anti-aging strategies. We therefore propose that stochastic biomathematical modeling and computational simulation platforms should be developed as in silico strategies to accelerate the performance of clinical trials targeting human aging, and to provide personalized approaches and robust biomarkers of healthy aging at the individual-to-population levels.

Background: The minor allele (C) of the single-nucleotide polymorphism (SNP) rs11212617, located near the ataxia telangiectasia mutated (ATM) gene, has been associated with an increased likelihood of treatment success with metformin in type 2 diabetes. We herein investigated whether the same SNP would predict clinical response to neoadjuvant metformin in women with early breast cancer (BC). Methods: DNA was collected from 79 patients included in the intention-to-treat population of the METTEN study, a phase 2 clinical trial of HER2-positive BC patients randomized to receive either metformin combined with anthracycline/taxane-based chemotherapy and trastuzumab or equivalent regimen without metformin, before surgery. SNP rs11212617 genotyping was assessed using allelic discrimination by quantitative polymerase chain reaction. Results: Logistic regression analyses revealed a significant relationship between the rs11212617 genotype and the ability of treatment arms to achieve a pathological complete response (pCR) in patients (odds ratio [OR]genotype×arm = 10.33, 95% confidence interval [CI]: 1.29–82.89, p = 0.028). In the metformin-containing arm, patients bearing the rs11212617 C allele had a significantly higher probability of pCR (ORA/C,C/C = 7.94, 95%CI: 1.60–39.42, p = 0.011). Conversely, no association was found between rs11212617 and clinical response in the reference arm (ORA/C,C/C = 0.77, 95%CI: 0.20–2.92, p = 0.700). After controlling for tumor size and hormone receptor status, the rs11212617 C allele remained a significant predictor of pCR solely in the metformin-containing arm. Conclusions: If reproducible, the rs11212617 C allele might warrant consideration as a predictive clinical biomarker to inform the personalized use of metformin in BC patients. Trial Registration: EU Clinical Trials Register, EudraCT number 2011-000490-30. Registered 28 February 2011, https://www.clinicaltrialsregister.eu/ctr-search/trial/2011-000490-30/ES.

Metformina; Ki67; Càncer de mama Metformina; Ki67; Cáncer de mama Metformin; Ki67; Breast cancer The proliferative capacity of residual breast cancer (BC) disease indicates the existence of partial treatment resistance and higher probability of tumor recurrence. We explored the therapeutic potential of adding neoadjuvant metformin as an innovative strategy to decrease the proliferative potential of residual BC cells in patients failing to achieve pathological complete response (pCR) after pre-operative therapy. We performed a prospective analysis involving the intention-to-treat population of the (Metformin and Trastuzumab in Neoadjuvancy) METTEN study, a randomized multicenter phase II trial of women with primary, non-metastatic (human epidermal growth factor receptor 2) HER2-positive BC evaluating the efficacy, tolerability, and safety of oral metformin (850 mg twice-daily) for 24 weeks combined with anthracycline/taxane-based chemotherapy and trastuzumab (arm A) or equivalent regimen without metformin (arm B), before surgery. We centrally evaluated the proliferation marker Ki67 on sequential core biopsies using visual assessment (VA) and an (Food and Drug Administration) FDA-cleared automated digital image analysis (ADIA) algorithm. ADIA-based pre-operative values of high Ki67 (≥20%), but not those from VA, significantly predicted the occurrence of pCR in both arms irrespective of the hormone receptor status (p = 0.024 and 0.120, respectively). Changes in Ki67 in residual tumors of non-pCR patients were significantly higher in the metformin-containing arm (p = 0.025), with half of all patients exhibiting high Ki67 at baseline moving into the low-Ki67 (