Joaquim Bosch-Barrera

Silibinin and STAT3: A natural way of targeting transcription factors for cancer therapy

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many different types of cancer and plays a pivotal role in tumor growth and metastasis. Retrospective studies have established that STAT3 expression or phospho-STAT3 (pSTAT3 or activated STAT3) are poor prognostic markers for breast, colon, prostate and non-small cell lung cancer. Silibinin or silybin is a natural polyphenolic flavonoid which is present in seed extracts of milk thistle (Silybum marianum). Silibinin has been shown to inhibit multiple cancer cell signaling pathways in preclinical models, demonstrating promising anticancer effects in vitro and in vivo. This review summarizes evidence suggesting that silibinin can inhibit pSTAT3 in preclinical cancer models. We also discuss current strategies to overcome the limitations of oral administration of silibinin to cancer patients to translate the bench results to the bed side. Finally, we review the ongoing clinical trials exploring the role of silibinin in cancer.

Metformin lowers the threshold for stress-induced senescence: A role for the microRNA-200 family and miR-205

We have tested the hypothesis that the antidiabetic biguanide metformin can be used to manipulate the threshold for stress-induced senescence (SIS), thus accelerating the onset of cancer-protective cellular senescence in response to oncogenic stimuli. Using senescence-prone murine embryonic fibroblasts (MEFs), we assessed whether metformin treatment modified the senescence phenotype that is activated in response to DNA damaging inducers. Metformin significantly enhanced the number of MEFs entering a senescent stage in response to doxorubicin, an anthracycline that induces cell senescence by activating DNA damage signaling pathways (e.g., ATM/ATR) in a reactive oxygen species (ROS)-dependent manner. Using WI-38 and BJ-1 human diploid fibroblasts (HDFs), we explored whether metformin supplementation throughout their entire replicative lifespan may promote the early appearance of the biomarkers of replicative senescence. Chronic metformin significantly reduced HDFs’ lifespan by accelerating both the loss of replicative potential and the acquisition of replicative senescence-related biomarkers (e.g., enlarged and flattened cell shapes, loss of arrayed arrangement, accumulation of intracellular and extracellular debris and SA-β-gal-positive staining). Metformin functioned as a bona fide stressful agent, inducing monotonic, dose-dependent, SIS-like responses in BJ-1 HDFs, which are highly resistant to ROS-induced premature senescence. Metformin-induced SIS in BJ-1 fibroblasts was accompanied by the striking activation of several microRNAs belonging to the miR-200s family (miR-200a, miR-141 and miR429) and miR-205, thus mimicking a recently described ability of ROS to chemosensitize cancer cells by specifically upregulating anti-EMT (epithelial-to-mesenchymal transition) miR-200s. Because the unlimited proliferative potential of stem cells results from their metabolic refractoriness to SIS, we finally tested if metformin treatment could circumvent the stress (e.g., ROS)-resistant phenotype of induced pluripotent stem cells (iPSCs). Metformin treatment drastically reduced both the number and the size of iPSC colonies and notably diminished the staining of the pluripotency marker alkaline phosphatase. Our current findings, altogether, reveal for the first time that metformin can efficiently lower the threshold for SIS to generate an “stressed” cell phenotype that becomes pre-sensitized to oncogenic-like stimuli, including DNA damaging, proliferative and/or stemness inducers.

Silibinin suppresses EMT-driven erlotinib resistance by reversing the high miR-21 /low miR-200c signature in vivo

The flavolignan silibinin was studied for its ability to restore drug sensitivity to EGFR-mutant NSCLC xenografts with epithelial-to-mesenchymal transition (EMT)-driven resistance to erlotinib. As a single agent, silibinin significantly decreased the tumor volumes of erlotinib-refractory NSCLC xenografts by approximately 50%. Furthermore, the complete abrogation of tumor growth was observed with the co-treatment of erlotinib and silibinin. Silibinin fully reversed the EMT-related high miR-21/low miR-200c microRNA signature and repressed the mesenchymal markers SNAIL, ZEB, and N-cadherin observed in erlotinib-refractory tumors. Silibinin was sufficient to fully activate a reciprocal mesenchymal-to-epithelial transition (MET) in erlotinib-refractory cells and prevent the highly migratogenic phenotype of erlotinib-resistant NSCLC cells. Given that the various mechanisms of resistance to erlotinib result from EMT, regardless of the EGFR mutation status, a water-soluble, silibinin-rich milk thistle extract might be a suitable candidate therapy for upcoming clinical trials aimed at preventing or reversing NSCLC progression following erlotinib treatment.

Stem cell-like ALDHbright cellular states in EGFR-mutant non-small cell lung cancer: A novel mechanism of acquired resistance to erlotinib targetable with the natural polyphenol silibinin

The enrichment of cancer stem cell (CSC)-like cellular states has not previously been considered to be a causative mechanism in the generalized progression of EGFR-mutant non-small cell lung carcinomas (NSCLC) after an initial response to the EGFR tyrosine kinase inhibitor erlotinib. To explore this possibility, we utilized a pre-clinical model of acquired erlotinib resistance established by growing NSCLC cells containing a TKI-sensitizing EGFR exon 19 deletion (ΔE746-A750) in the continuous presence of high doses of erlotinib. Genome-wide analyses using Agilent 44K Whole Human Genome Arrays were evaluated via bioinformatics analyses through GSEA-based screening of the KEGG pathway database to identify the molecular circuitries that were over-represented in the transcriptomic signatures of erlotinib-refractory cells. The genomic spaces related to erlotinib resistance included a preponderance of cell cycle genes (E2F1, -2, CDC2, -6) and DNA replication-related genes (MCM4, -5, -6, -7), most of which are associated with early lung development and poor prognosis. In addition, metabolic genes such as ALDH1A3 (a candidate marker for lung cancer cells with CSC-like properties) were identified. Thus, we measured the proportion of erlotinib-resistant cells expressing very high levels of aldehyde dehydrogenase (ALDH) activity attributed to ALDH1/3 isoforms. Using flow cytometry and the ALDEFLUOR® reagent, we confirmed that erlotinib-refractory cell populations contained drastically higher percentages (>4500%) of ALDHbright cells than the parental erlotinib-responsive cells. Notably, strong decreases in the percentages of ALDHbright cells were observed following incubation with silibinin, a bioactive flavonolignan that can circumvent erlotinib resistance in vivo. The number of lung cancer spheres was drastically suppressed by silibinin in a dose-dependent manner, thus confirming the ability of this agent to inhibit the self-renewal of erlotinib-refractory CSC-like cells. This report is the first to show that: (1) loss of responsiveness to erlotinib in EGFR-mutant NSCLC can be explained in terms of erlotinib-refractory ALDHbright cells, which have been shown to exhibit stem cell-like properties; and (2) erlotinib-refractory ALDHbright cells are sensitive to the natural agent silibinin. Our findings highlight the benefit of administration of silibinin in combination with EGFR TKIs to target CSCs and minimize the ability of tumor cells to escape cell death in EGFR-mutant NSCLC patients.

Metformin rescues cell surface major histocompatibility complex class I (MHC-I) deficiency caused by oncogenic transformation.

Active avoidance by tumor cells from attack and elimination by immune cells is an emerging cancer hallmark that is achieved primarily through decreasing the levels of major histocompatibility complex class I (MHC-I) at the cancer cells’ surface. Deficiencies in MHC-I antigen-restricted immunosurveillance may be intertwined with an altered, Warburg-like cancer cell-intrinsic metabolism, another emerging hallmark of cancer that involves a switch from mitochondrial respiration to glycolysis to efficiently support large-scale biosynthetic programs that are required for active cell proliferation. We recently envisioned that intervention strategies aimed at reversing the bioenergetic signature of cancer cells (e.g., the antidiabetic biguanide metformin) should correct oncogene (e.g., HER2)-driven MHC-I defects, thus preventing immune escape of oncogene transformants. First, we explored how metformin treatment impacted mitochondrial biogenesis in cultured breast cancer cells overexpressing the membrane tyrosine kinase receptor HER2, the best-characterized downregulator of MHC-I. Metformin exposure was found to dose-dependently increase the expression levels of cytochrome c oxidase I and mitochondrial succinate dehydrogenase, which are encoded by mitochondrial and nuclear DNA, respectively. Second, we explored whether metformin-enhanced mitochondrial biogenesis might significantly alter the MHC-I status in breast carcinoma cells. MHC-I expression, as assessed by flow cytometry using an anti-HLA-ABC monoclonal antibody, was fully restored (up to ~25-fold upregulation) in MHC-I-negative HER2 gene-amplified carcinoma cells. These findings may help delineate a previously unrecognized mechanism through which metformin (and metformin-like drugs) may enable a cancer patient’s own immune system to mount an efficient anti-metastasis response that can prevent or delay disease recurrence. Restored antigenicity and immunogenicity of tumor cells may represent a previously unrecognized primary mode of action underlying the cancer-preventive effects of metformin.

Cross-suppression of EGFR ligands amphiregulin and epiregulin and de-repression of FGFR3 signalling contribute to cetuximab resistance in wild-type KRAS tumour cells.

BACKGROUND:In addition to the mutational status of KRAS, the epidermal growth factor receptor (EGFR) ligands amphiregulin (AREG) and epiregulin (EREG) might function as bona fide biomarkers of cetuximab (Ctx) sensitivity for most EGFR-driven carcinomas.METHODS:Lentivirus-delivered small hairpin RNAs were employed to specifically reduce AREG or EREG gene expression in wild-type KRAS A431 squamous cell carcinoma cells. Colony-forming assays were used to monitor the impact of AREG and EREG knockdown on Ctx efficacy. Amphiregulin and EREG protein expression levels were assessed by quantitative ELISA in parental A431 cells and in pooled populations of A431 cells adapted to grow in the presence of Ctx. A phosphoproteomic platform was used to measure the relative level of phosphorylation of 42 distinct receptor tyrosine kinases before and after the acquisition of resistance to Ctx.RESULTS:Stable gene silencing of either ligand was found to notably reduce the expression of the other ligand. Parental A431 cells with normal expression levels of AREG/EREG exhibited significantly increased growth inhibition in response to Ctx, compared with derivatives that are engineered to produce minimal AREG/EREG. The parental A431 cells acutely treated with Ctx exhibited reduced basal expression levels of AREG/EREG. Pooled populations of Ctx-resistant A431 cells expressed significantly lower levels of AREG/EREG and were insensitive to the downregulatory effects of Ctx. Phosphoproteomic screen identified a remarkable hyperactivation of FGFR3 in Ctx-resistant A431 cells, which gained sensitivity to the cytotoxic and apoptotic effects of the FGFR3 TK inhibitor PD173074. The A431 parental cells acutely treated with Ctx rapidly activated FGFR3 and their concomitant exposure to Ctx and PD173074 resulted in synergistic apoptosis.CONCLUSION:Cross-suppression of AREG/EREG expression may explain the tight co-expression of AREG and EREG, as well as their tendency to be more highly expressed than other EGFR ligands to determine Ctx efficacy. The positive selection for Ctx-resistant tumour cells exhibiting AREG/EREG cross-suppression may have an important role in the emergence of Ctx resistance. As de-repression of FGFR3 activity rapidly replaces the loss of EGFR-ligand signalling in terms of cell proliferation and survival, combinations of Ctx and FGFR3-targeted drugs may be a valuable strategy to enhance the efficacy of single Ctx while preventing or delaying acquired resistance to Ctx.British Journal of Cancer (2012) 106, 1406–1414. doi:10.1038/bjc.2012.103 www.bjcancer.com

Silibinin meglumine, a water-soluble form of milk thistle silymarin, is an orally active anti-cancer agent that impedes the epithelial-to-mesenchymal transition (EMT) in EGFR-mutant non-small-cell lung carcinoma cells

Silibinin is the primary active constituent of a crude extract (silymarin) from milk thistle plant (Silybum marianum) seeds. We explored the ability of an oral milk thistle extract formulation that was enriched with a water-soluble form of silibinin complexed with the amino-sugar meglumine to inhibit the growth of non-small-cell lung carcinoma (NSCLC) mouse xenografts. As a single agent, oral silibinin meglumine notably decreased the overall volumes of NSCLC tumors as efficiently as did the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Concurrent treatment with silibinin meglumine impeded the regrowth of gefitinib-unresponsive tumors, resulting in drastic tumor growth prevention. Because the epithelial-to-mesenchymal transition (EMT) is required by a multiplicity of mechanisms of resistance to EGFR TKIs, we evaluated the ability of silibinin meglumine to impede the EMT in vitro and in vivo. Silibinin-meglumine efficiently prevented the loss of markers associated with a polarized epithelial phenotype as well as the de novo synthesis of proteins associated with the mesenchymal morphology of transitioning cells. Our current findings with this non-toxic, orally active, and water-soluble silibinin formulation might facilitate the design of clinical trials to test the administration of silibinin meglumine-containing injections, granules, or beverages in combination with EGFR TKIs in patients with EGFR-mutated NSCLC.

Id1 and Id3 co-expression correlates with clinical outcome in stage III-N2 non-small cell lung cancer patients treated with definitive chemoradiotherapy

BackgroundInhibitor of DNA binding 1 (Id1) and 3 (Id3) genes have been related with the inhibition of cell differentiation, cell growth promotion and tumor metastasis. Recently, Id1 has been identified as an independent prognostic factor in patients with lung adenocarcinoma, regardless of the stage. Furthermore, Id1 may confer resistance to treatment (both, radiotherapy and chemotherapy).MethodsWe have studied, using monoclonal antibodies for immunohistochemistry, the Id1 and Id3 tumor epithelial expression in 17 patients with stage III-N2 non-small cell lung cancer (NSCLC) treated with definitive chemoradiotherapy.ResultsId1 expression is observed in 82.4% of the tumors, whereas Id3 expression is present in 41.2% of the samples. Interestingly, Id1 and Id3 expression are mutually correlated (R = 0.579, p = 0.015). In a subgroup analysis of patients with the most locally advanced disease (T4N2 stage), co-expression of Id1 and Id3 showed to be related with a worse overall survival (45 vs 6 months, p = 0.002). A trend towards significance for a worse progression free survival (30 vs 1 months, p = 0.219) and a lower response rate to the treatment (RR = 50% vs 87.5%, p = 0.07) were also observed.ConclusionsA correlation between Id1 and Id3 protein expression is observed. Id1 and Id3 co-expression seems associated with a poor clinical outcome in patients with locally advanced NSCLC treated with definitive chemoradiotherapy.

Toxic epidermal necrolysis related to pemetrexed and carboplatin with vitamin B12 and folic acid supplementation for advanced non-small cell lung cancer.

Background: Pemetrexed is a multitargeted antifolate initially approved as a single agent for the second-line treatment of locally advanced or metastatic non-small cell lung cancer and more recently in the first-line setting combined with cisplatin. The combination of pemetrexed with carboplatin has been tested in several phase II clinical trials showing interesting antitumour activity with mild toxicity. Supplementation with folic acid and vitamin B12 during treatment with pemetrexed is recommended to reduce potential haematological and gastrointestinal adverse events. Case Report: A patient experienced cutaneous lesions including widespread erythema, epidermal detachment, and skin denudation, associated with deterioration of his general condition after the second cycle of this chemotherapy combination, which was clinically and histologically compatible with toxic epidermal necrolysis (Lyell’s syndrome). Treatment with systemic steroids, antihistamines, and antibiotics led to resolution of the skin lesions and improvement of his general condition. Conclusion: To our knowledge, this is the second case reported in the literature of this type of suspected adverse drug reaction associated with a pemetrexed-based chemotherapy combination.

BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer

BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.