Highly Sensitive Microsatellite Instability and Immunohistochemistry Assessment in Endometrial Aspirates as a Tool for Cancer Risk Individualization in Lynch Syndrome.

Women with Lynch syndrome (LS) are at increased risk of endometrial cancer (EC), among other tumors, and are characterized by mismatch repair (MMR) deficiency and microsatellite instability (MSI). While risk-reducing gynecologic surgeries effectively decrease EC incidence, doubts arise regarding the appropriate timing of the surgery. We explored the usefulness of highly sensitive MSI (hs-MSI) assessment in endometrial aspirates for individualizing gynecologic surveillance in LS carriers. Ninety-three women with LS, 25 sporadic EC patients (9 MMR-proficient and 16 MMR-deficient), and 30 women with benign gynecologic disease were included in this study. hs-MSI was assessed in prospectively collected endometrial aspirates in 67 LS carriers, EC cases, and controls. MMR, PTEN, ARID1A, and PAX2 protein expression patterns were evaluated in the LS samples. Follow-up aspirates from 8 LS carriers were also analyzed. Elevated hs-MSI scores were detected in all aspirates from MMR-deficient EC cases (3 LS and 16 sporadic) and negative in aspirates from controls and MMR-proficient EC cases. Positive hs-MSI scores were also detected in all 4 LS aspirates reported as complex hyperplasia. High hs-MSI was also present in 10 of 49 aspirates (20%) from LS carriers presenting a morphologically normal endometrium, where MMR protein expression loss was detected in 69% of the samples. Interestingly, the hs-MSI score was positively correlated with MMR-deficient gland density and the presence of MMR-deficient clusters, colocalizing PTEN and ARID1A expression loss. High hs-MSI scores and clonality were evidenced in 2 samples collected up to 4 months before EC diagnosis; hs-MSI scores increased over time in 5 LS carriers, whereas they decreased in a patient with endometrial hyperplasia after progestin therapy. In LS carriers, elevated hs-MSI scores were detected in aspirates from premalignant and malignant lesions and normal endometrium, correlating with MMR protein loss. hs-MSI assessment and MMR immunohistochemistry may help individualize EC risk assessment in women with LS.

Epidemiology and Molecular Profile of Mucosal Melanoma: A Population-Based Study in Southern Europe.

BACKGROUND: Mucosal melanoma is a rare neoplasm on which few epidemiological population-based studies have been published. A good surgical approach is the standard treatment, but the prognosis is worse than that of skin melanoma. The analysis of mucosal melanoma's mutational profile can help to develop target therapies in advanced disease or adjuvant settings. METHODS: We analyzed the database of the Cancer Registry of Girona, a region located in the north-east of Spain, in the period of 1994-2018. We selected cases of primary invasive melanoma, excluding those located in the skin, eye, central nervous system and an unknown primary site. Epidemiological analysis included incidence and survival. Mutational profile analysis was performed with a custom gene panel. RESULTS: Forty-two patients were identified: 14 (33%) had vulvar-vaginal melanoma, 15 (35.7%) had rectal melanoma, 12 (28.6%) had melanoma located in the head and neck sphere and 1 male patient had a urethral melanoma. European age-standardized incidence rates for vulvar-vaginal, rectal and head and neck melanoma were 0.09, 0.1 and 0.09 cases/100,000 inhabitant-years, respectively. Five-year observed survival rates were 37.5%, 20% and 25% for these types of cancers. NRAS Q61 was the most frequent mutation found. CONCLUSION: Our study confirms the steady incidence and low survival of mucosal melanomas in a region of southern Europe. NRAS and NF1 play a role in the molecular landscape of mucosal melanoma. MEK and PI3K/mTOR inhibitors could be reasonable treatment options and are being studied in clinical trials.

Population-based analysis of the prevalence of BRAF mutation in patients diagnosed with cutaneous melanoma and its significance as a prognostic factor.

The prevalence of BRAF mutation has been reported in between 38% and 48% of melanoma patients, based on mainly Stage III or metastatic melanoma, however, information based on population-based studies is scarce. We performed a population-based retrospective cohort study to determine the prevalence of the BRAF mutation in patients diagnosed with in situ and infiltrating cutaneous malignant melanoma in the province of Girona between 2009 and 2011. Using the database of the Girona Cancer Registry, we performed BRAF mutation analysis based on paraffin-embedded tissue. This data was then correlated with other known clinical and histological prognostic factors for survival. We found 286 incident cases of cutaneous melanoma in the Girona Cancer Registry database. Excluding missing cases, BRAF-mutated patients constituted 38.9% of "in situ" melanoma cases and 53.8% of invasive melanoma cases. Five-year relative survival was not statistically different between BRAF-mutated patients (93.6%; 95% CI: 87.1-100.5) and non-mutated patients (84.3%, 95% CI: 75.3-94.8). Only stage was significant as a prognostic factor for survival based on multivariate analysis. From our population-based study, we conclude that BRAF mutation is not an independent prognostic factor for melanoma survival.

Silibinin Suppresses Tumor Cell-Intrinsic Resistance to Nintedanib and Enhances Its Clinical Activity in Lung Cancer.

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.

Long-term protection of HPV test in women at risk of cervical cancer.

OBJECTIVE: To evaluate the 9-year incidence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and cumulative adherence to perform a next test in a cohort of women aged 40+ years with no cervical screening cytology within a window of 5 years (underscreened women), after baseline cervical cytology and HPV tests. METHODS: In Catalonia, Spain, co-testing with cytology and HPV test has been recommended in the Public Health system since 2006 for underscreened women. In 2007, 1,594 women with underscreened criteria were identified and followed through medical records form Pathological Department. 9-year cumulative incidence of histologically confirmed CIN2+ and cumulative adherence to perform a next test were estimated using Kaplan-Meier statistics. RESULTS: Follow-up was available for 1,009 women (63.3%) resulting in 23 women with. CIN2+ (2.3%). Of them, 4 women (17%) had both tests negative at baseline (3CIN2 and 1CIN3) with cumulative incidence of CIN2+ of 0.4% (95% CI: 0.1-1.4) at 5-years and 1.3% (95% CI: 0.4-3.7) at 9-years. During the first year, the prevalence among women with both tests positive was 27.0% (95% CI: 13.0-50.6) for CIN2+. Lost to follow-up was higher among women with both tests negative compared to those with both positive tests (38.7% vs 4.2%, p-value <0.001). 40.5% of the women HPV-/cyto- had a re-screening test during the 4 years following the baseline, increasing until 53.5% during the 6 years of follow-up. CONCLUSIONS: HPV detection shows a high longitudinal predictive value at 9-year to identify women at risk to develop CIN2+. The data validate a safe extension of the 3-year screening intervals (current screening interval) to 5-year intervals in underscreened women that had negative HPV result at baseline. It is necessary to establish mechanisms to ensure screening participation and adequate follow-up for these women.

Neoadjuvant Metformin Added to Systemic Therapy Decreases the Proliferative Capacity of Residual 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 (<20%) category after neoadjuvant treatment. By contrast, no statistically significant changes in Ki67 occurred in residual tumors of the control treatment arm (p = 0.293). There is an urgent need for innovative therapeutic strategies aiming to provide the protective effects of decreasing Ki67 after neoadjuvant treatment even if pCR is not achieved. Metformin would be evaluated as a safe candidate to decrease the aggressiveness of residual disease after neoadjuvant (pre-operative) systemic therapy of BC patients.

Highly sensitive MLH1 methylation analysis in blood identifies a cancer patient with low-level mosaic MLH1 epimutation.

Constitutional MLH1 methylation (epimutation) is a rare cause of Lynch syndrome. Low-level methylation (≤ 10%) has occasionally been described. This study aimed to identify low-level constitutional MLH1 epimutations and determine its causal role in patients with MLH1-hypermethylated colorectal cancer.Eighteen patients with MLH1-hypermethylated colorectal tumors in whom MLH1 methylation was previously undetected in blood by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were screened for MLH1 methylation using highly sensitive MS-melting curve analysis (MS-MCA). Constitutional methylation was characterized by different approaches.MS-MCA identified one patient (5.6%) with low-level MLH1 methylation (~ 1%) in blood and other normal tissues, which was confirmed by clonal bisulfite sequencing in blood. The patient had developed three clonally related gastrointestinal MLH1-methylated tumor lesions at 22, 24, and 25 years of age. The methylated region in normal tissues overlapped with that reported for other carriers of constitutional MLH1 epimutations. Low-level MLH1 methylation and reduced allelic expression were linked to the same genetic haplotype, whereas the opposite allele was lost in patient's tumors. Mutation screening of MLH1 and other hereditary cancer genes was negative.Herein, a highly sensitive MS-MCA-based approach has demonstrated its utility for the identification of low-level constitutional MLH1 epigenetic mosaicism. The eventual identification and characterization of additional cases will be critical to ascertain the cancer risks associated with constitutional MLH1 epigenetic mosaicism.

Epigenetic silencing of TGFBI confers resistance to trastuzumab in human breast cancer.

BACKGROUND: Acquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients. METHODS: We performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing. RESULTS: Epigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients. CONCLUSIONS: Our results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Next-generation Sequencing for ALK and ROS1 Rearrangement Detection in Patients With Non-small-cell Lung Cancer: Implications of FISH-positive Patterns.

BACKGROUND: Detection of ALK and ROS1 gene rearrangements in non-small-cell lung cancer is required for directing patient care. Although fluorescence in situ hybridization (FISH) and immunohistochemistry have been established as gold standard methods, next-generation sequencing (NGS) platforms are called to be at least equally successful. Comparison of these methods for translation into daily use is currently under investigation. PATIENTS AND METHODS: Forty non-small-cell lung cancer paraffin-embedded samples with previous ALK (n = 33) and ROS1 (n = 7) FISH results were examined with the Oncomine Focus Assay and tested for ALK and ROS1 immunoreactivity. Clinical implications of concurrent molecular alterations and concordance between methods were evaluated. RESULTS: NGS was successful in 32 (80%) cases: 25 ALK and 7 ROS1. Few concomitant alterations were detected: 1 ALK rearranged case had an ALK p.L1196M-resistant mutation, 4 had CDK4, MYC, and/or ALK amplifications, and 1 ROS1 rearranged case showed a FGFR4 amplification. Comparison between techniques revealed 5 (16%) discordant cases that had lower progression-free survival than concordant cases: 7.6 (95% confidence interval, 2.2-13) versus 19.4 (95% confidence interval, 10.1-28.6). Remarkably, 4 of these cases had isolated 3' signal FISH pattern (P = .026). CONCLUSION: Our data support that the identification of 3' isolated signal FISH pattern in ALK and ROS1 cases might suggest a false-positive result. NGS seems a reliable technique to assess ALK and ROS1 rearrangements, offering the advantage over immunohistochemistry of detecting other molecular alterations with potential therapeutic implications.

Fatty acid synthase expression and its association with clinico-histopathological features in triple-negative breast cancer.

Triple-Negative Breast Cancer (TNBC) has poor prognosis and no approved targeted therapy. We previously showed that the enzyme fatty acid synthase (FASN) was largely expressed in a small TNBC patients' cohort and its inhibition synergized with cetuximab in TNBC preclinical mouse models. Here, we evaluated FASN and EGFR expression in a cohort of TNBC patients and we study their prognostic role and their association with clinico-histopathological features, intrinsic TNBC subtypes and survival. FASN, EGFR, CK5/6 and vimentin expression were retrospective evaluated by Immunohistochemistry in 100 primary TNBC tumors. FASN expression was classified into high and low FASN groups. EGFR, CK5/6 and vimentin expression were used in TNBC intrinsic subtypes classification. FASN was expressed in most of the TNBC patients but did not correlate with overall survival or disease-free survival in this cohort. High FASN group was significantly associated with positive node status. FASN expression was significantly higher in Basal-Like patients than in Mesenchymal-Like ones. EGFR expression was positive in 50% of the tumors, and those patients showed poorer DFS. Altogether, our findings provide a rationale for further investigation the prognostic role of FASN and EGFR expression in a larger cohort of TNBC patients.

Preclinical Evaluation of Fatty Acid Synthase and EGFR Inhibition in Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) lacks an approved targeted therapy. Despite initial good response to chemotherapy, 30% of the patients relapse within 5 years after treatment. EGFR overexpression is a common marker in TNBC, and its expression has been correlated with poor outcome. Inhibition of fatty acid synthase (FASN) activity leads to apoptosis of human carcinoma cells overexpressing FASN. We tested the hypothesis that blocking FASN in combination with anti-EGFR signaling agents would be an effective antitumor strategy in sensitive and chemoresistant TNBC. EXPERIMENTAL DESIGN: Several TNBC cell lines and 29 primary tumors were included to determine whether FASN is a potential target in TNBC. Doxorubicin-resistant TNBC cell lines (231DXR and HCCDXR) have been developed and characterized in our laboratory. Cellular and molecular interactions of anti-FASN compounds (EGCG and C75) with cetuximab were analyzed. In vivo tumor growth inhibition was evaluated after cetuximab, EGCG, or the combination in TNBC orthoxenograft models. RESULTS: TNBC cell lines showed overexpression of FASN enzyme and its inhibition correlated to FASN levels. FASN staining was observed in all of the 29 TNBC tumor samples. In vitro, EGCG and C75 plus cetuximab showed strong synergism in sensitive and chemoresistant cells. In vivo, the combination of EGCG with cetuximab displayed strong antitumor activity against the sensitive and chemoresistant TNBC orthoxenografts, without signs of toxicity. CONCLUSIONS: Our results show that the simultaneous blockade of FASN and EGFR is effective in preclinical models of sensitive and chemoresistant TNBC. Clin Cancer Res; 22(18); 4687-97. ©2016 AACR.

Soluble CRTC3: A Newly Identified Protein Released by Adipose Tissue That Is Associated with Childhood Obesity.

BACKGROUND: CREB-regulated transcription coactivator 3 (CRTC3) is found in adipocytes, where it may promote obesity through disruption of catecholamine signaling. We wished to assess whether CRTC3 is a soluble protein secreted by adipose tissue, explore whether CRTC3 is detectable and quantifiable in the circulation, and ascertain whether CRTC3 serum concentrations are related to metabolic markers in children. METHODS: Explants of adipose tissue from 12 children were cultured to study adipocyte cell size and the secretion of CRTC3 (immunoblot and ELISA). We also performed a cross-sectional and longitudinal study in 211 asymptomatic prepubertal white children at age 7 years, 115 of whom were followed up at age approximately 10 years. We measured circulating concentrations of CRTC3 and studied associations between serum CRTC3 and metabolic markers. RESULTS: Measurable concentrations of CRTC3 were found in conditioned media of adipose tissue explants and in serum samples. CRTC3 concentrations in visceral adipose tissue were negatively associated with adipocyte cell size and positively related to adipocyte cell number (P < 0.05). In the cross-sectional study, higher CRTC3 concentrations were associated with higher body mass index (P = 0.001), waist circumference (P = 0.003), and systolic blood pressure (P = 0.007) and lower high molecular weight adiponectin (P = 0.003). In the longitudinal study, serum concentrations of CRTC3 at age approximately 7 years were associated with changes in waist circumference (ß = 0.254; P = 0.004; r = 0.145) and high molecular weight adiponectin (ß=-0.271; P = 0.014; r = 0.101), respectively, at age approximately 10 years. CONCLUSIONS: CRTC3, a newly identified protein that is related to childhood obesity, is present in the circulation, partly as a result of adipose tissue secretion. Higher serum CRTC3 concentrations are related to and predict a poorer metabolic profile in children.

Dual fatty acid synthase and HER2 signaling blockade shows marked antitumor activity against breast cancer models resistant to anti-HER2 drugs.

Blocking the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of HER2-positive breast carcinoma cells. The hypothesis is that blocking FASN, in combination with anti-HER2 signaling agents, would be an effective antitumor strategy in preclinical HER2+ breast cancer models of trastuzumab and lapatinib resistance. We developed and molecularly characterized in vitro HER2+ models of resistance to trastuzumab (SKTR), lapatinib (SKLR) and both (SKLTR). The cellular interactions of combining anti-FASN polyphenolic compounds (EGCG and the synthetic G28UCM) with anti-HER2 signaling drugs (trastuzumab plus pertuzumab and temsirolimus) were analyzed. Tumor growth inhibition after treatment with EGCG, pertuzumab, temsirolimus or the combination was evaluated in two in vivo orthoxenopatients: one derived from a HER2+ patient and another from a patient who relapsed on trastuzumab and lapatinib-based therapy. SKTR, SKLR and SKLTR showed hyperactivation of EGFR and p-ERK1/2 and PI3KCA mutations. Dual-resistant cells (SKLTR) also showed hyperactivation of HER4 and recovered levels of p-AKT compared with mono-resistant cells. mTOR, p-mTOR and FASN expression remained stable in SKTR, SKLR and SKLTR. In vitro, anti-FASN compounds plus pertuzumab showed synergistic interactions in lapatinib- and dual- resistant cells and improved the results of pertuzumab plus trastuzumab co-treatment. FASN inhibitors combined with temsirolimus displayed the strongest synergistic interactions in resistant cells. In vivo, both orthoxenopatients showed strong response to the antitumor activity of the combination of EGCG with pertuzumab or temsirolimus, without signs of toxicity. We showed that the simultaneous blockade of FASN and HER2 pathways is effective in cells and in breast cancer models refractory to anti-HER2 therapies.

Fatty acid synthase expression is strongly related to menopause in early-stage breast cancer patients.

OBJECTIVE: Overexpression of fatty acid synthase (FASN), the enzyme involved in the de novo synthesis of fatty acids, has been reported in several human carcinomas, including breast cancer, and has been related to poor prognosis. Our aim was to analyze the association of FASN tumor tissue expression with clinicopathological and anthropometrical features in early-stage breast cancer patients. METHODS: We prospectively studied 53 women with early-stage breast cancer who were treated with surgical operation and postoperative chemotherapy. RESULTS: Menopause status and age were strongly associated with higher levels of FASN tumor expression (P < 0.005 and P = 0.038, respectively). Body mass index and pathological stage were also related to FASN tumor expression. CONCLUSIONS: Our findings suggest that FASN could be a potential therapeutic target in postmenopausal breast cancer patients. However, further studies are needed.

Different fatty acid metabolism effects of (-)-epigallocatechin-3-gallate and C75 in adenocarcinoma lung cancer.

BACKGROUND: Fatty acid synthase (FASN) is overexpressed and hyperactivated in several human carcinomas, including lung cancer. We characterize and compare the anti-cancer effects of the FASN inhibitors C75 and (-)-epigallocatechin-3-gallate (EGCG) in a lung cancer model. METHODS: We evaluated in vitro the effects of C75 and EGCG on fatty acid metabolism (FASN and CPT enzymes), cellular proliferation, apoptosis and cell signaling (EGFR, ERK1/2, AKT and mTOR) in human A549 lung carcinoma cells. In vivo, we evaluated their anti-tumour activity and their effect on body weight in a mice model of human adenocarcinoma xenograft. RESULTS: C75 and EGCG had comparable effects in blocking FASN activity (96,9% and 89,3% of inhibition, respectively). In contrast, EGCG had either no significant effect in CPT activity, the rate-limiting enzyme of fatty acid ß-oxidation, while C75 stimulated CPT up to 130%. Treating lung cancer cells with EGCG or C75 induced apoptosis and affected EGFR-signaling. While EGCG abolished p-EGFR, p-AKT, p-ERK1/2 and p-mTOR, C75 was less active in decreasing the levels of EGFR and p-AKT. In vivo, EGCG and C75 blocked the growth of lung cancer xenografts but C75 treatment, not EGCG, caused a marked animal weight loss. CONCLUSIONS: In lung cancer, inhibition of FASN using EGCG can be achieved without parallel stimulation of fatty acid oxidation and this effect is related mainly to EGFR signaling pathway. EGCG reduce the growth of adenocarcinoma human lung cancer xenografts without inducing body weight loss. Taken together, EGCG may be a candidate for future pre-clinical development.

Androgen-independent prostate cancer cells circumvent EGFR inhibition by overexpression of alternative HER receptors and ligands.

The deregulation of the epidermal growth factor receptor (EGFR) pathway plays a major role in the pathogenesis of prostate cancer (PCa). However, therapies targeting EGFR have demonstrated limited effectiveness in PCa. A potential mechanism to overcome EGFR blockade in cancer cells is the autocrine activation of alternative receptors of the human EGFR (HER) family through the overexpression of the HER receptors and ligands. In the present study, we were interested in analyzing if this intrinsic resistance mechanism might contribute to the inefficacy of EGFR inhibitors in PCa. To this end, we selected two androgen-independent human prostate carcinoma cell lines (DU145 and PC3) and established DU145 erlotinib-resistant cells (DUErR). Cells were treated with three EGFR inhibitors (cetuximab, gefinitib and erlotinib) and the sensitivity to each treatment was assessed. The gene expression of the four EGFR/HER receptors and seven ligands of the HER family was analyzed by real-time PCR prior to and after each treatment. The receptors expression and activation were further characterized by flow cytometry and western blot analysis. EGFR inhibition rapidly induced enhanced gene expression of the EGF, betacellulin and neuregulin-1 ligands along with HER2, HER3 and HER4 receptors in the DU145 cells. In contrast, slight changes were observed in the PC3 cells, which are defective in the phosphatase and tensin homolog (PTEN) tumor suppressor gene. In the erlotinib-resistant DUErR cells, the expression of HER2 and HER3 was increased at mRNA and protein levels together with neuregulin-1, leading to enhanced HER3 phosphorylation and the activation of the downstream PI3K/Akt survival pathway. HER3 blockage by a monoclonal antibody restored the cytostatic activity of erlotinib in DUErR cells. Our results confirm that the overexpression and autocrine activation of HER3 play a key role in mediating the resistance to EGFR inhibitors in androgen-independent PCa cells.

New synthetic inhibitors of fatty acid synthase with anticancer activity.

Fatty acid synthase (FASN) is a lipogenic enzyme that is highly expressed in different human cancers. Here we report the development of a new series of polyphenolic compounds 5-30 that have been evaluated for their cytotoxic capacity in SK-Br3 cells, a human breast cancer cell line with high FASN expression. The compounds with an IC(50) < 50 µM have been tested for their ability to inhibit FASN activity. Among them, derivative 30 blocks the 90% of FASN activity at low concentration (4 µM), is highly cytotoxic in a broad panel of tumor cells, induces apoptosis, and blocks the activation of HER2, AKT, and ERK pathways. Remarkably, 30 does not activate carnitine palmitoyltransferase-1 (CPT-1) nor induces in mice weight loss, which are the main drawbacks of other previously described FASN inhibitors. Thus, FASN inhibitor 30 may aid the validation of this enzyme as a therapeutic target for the treatment of cancer.

A novel inhibitor of fatty acid synthase shows activity against HER2+ breast cancer xenografts and is active in anti-HER2 drug-resistant cell lines.

INTRODUCTION: Inhibiting the enzyme Fatty Acid Synthase (FASN) leads to apoptosis of breast carcinoma cells, and this is linked to human epidermal growth factor receptor 2 (HER2) signaling pathways in models of simultaneous expression of FASN and HER2. METHODS: In a xenograft model of breast carcinoma cells that are FASN+ and HER2+, we have characterised the anticancer activity and the toxicity profile of G28UCM, the lead compound of a novel family of synthetic FASN inhibitors. In vitro, we analysed the cellular and molecular interactions of combining G28UCM with anti-HER drugs. Finally, we tested the cytotoxic ability of G28UCM on breast cancer cells resistant to trastuzumab or lapatinib, that we developed in our laboratory. RESULTS: In vivo, G28UCM reduced the size of 5 out of 14 established xenografts. In the responding tumours, we observed inhibition of FASN activity, cleavage of poly-ADPribose polymerase (PARP) and a decrease of p-HER2, p- protein kinase B (AKT) and p-ERK1/2, which were not observed in the nonresponding tumours. In the G28UCM-treated animals, no significant toxicities occurred, and weight loss was not observed. In vitro, G28UCM showed marked synergistic interactions with trastuzumab, lapatinib, erlotinib or gefitinib (but not with cetuximab), which correlated with increases in apoptosis and with decreases in the activation of HER2, extracellular signal-regulated kinase (ERK)1/2 and AKT. In trastuzumab-resistant and in lapatinib-resistant breast cancer cells, in which trastuzumab and lapatinib were not effective, G28UCM retained the anticancer activity observed in the parental cells. CONCLUSIONS: G28UCM inhibits fatty acid synthase (FASN) activity and the growth of breast carcinoma xenografts in vivo, and is active in cells with acquired resistance to anti-HER2 drugs, which make it a candidate for further pre-clinical development.

Antimicrobial cyclic decapeptides with anticancer activity.

Antimicrobial peptides have been considered as potential candidates for cancer therapy. We report here the cytotoxicity of a library of 66 antibacterial cyclodecapeptides on human carcinoma cell lines, and their effects on apoptosis [as assessed by cleavage of poly(ADP-ribose) polymerase (PARP)] and cell signaling proteins (p53 and ERK1/2) in cultured human cervical carcinoma cells. A design of experiments approach permitted to analyze the results of a subset of 16 peptides and define rules for high anticancer activity against MDA-MB-231 breast carcinoma cells. Eight peptides were identified with IC(50) values ranging from 18.5 to 57.5 µM against the five cell lines tested, being HeLa cells the most sensitive. Among these sequences, BPC88, BPC96, BPC98, and BPC194 displayed specificity and high cytotoxicity against HeLa cells (IC(50) of 22.5-38.5 µM), showed low hemolytic activity and low cytotoxicity to non-malignant fibroblasts, and were stable to proteases in human serum. Induction of apoptosis by these peptides was observed and the apoptotic effect of BPC88 and BPC96 caused a marked decrease on the activated form of ERK1/2 kinase and an induction of p53. We further showed that BPC96 at low doses synergized the cytotoxic effect of cisplatin. These findings suggest that cyclic decapeptides may represent novel anticancer agents providing a new strategy in cancer therapy.

Activated nuclear factor kappa B and airway inflammation after smoke inhalation and burn injury in sheep.

In a recent study, we have shown a rapid inflammatory cell influx across the glandular epithelium and strong proinflammatory cytokine expression at 4 hours after inhalation injury. Studies have demonstrated a significant role of nuclear factor kappa B in proinflammatory cytokine gene transcription. This study examines the acute airway inflammatory response and immunohistochemical detection of p65, a marker of nuclear factor kappa B activation, in sheep after smoke inhalation and burn injury. Pulmonary tissue from uninjured sheep and sheep at 4, 8, 12, 24, and 48 hours after inhalation and burn injury was included in the study. Following immunostaining for p65 and myeloperoxidase, the cell types and the percentage of bronchial submucosal gland cells staining for p65 and the extent of myeloperoxidase stained neutrophils in the bronchial submucosa were determined. Results indicate absence of detection of P65 before 12 hours after injury. At 12 hours after injury, strong perinuclear staining for p65 was evident in bronchial gland epithelial cells, macrophages, and endothelial cells. Bronchial submucosal gland cells showed a significant increase in the percentage of cells stained for p65 compared with uninjured animals and earlier times after injury, P < .05. At 24 and 48 hours after injury, p65 expression was evident in the bronchiolar epithelium, Type II pneumocytes, macrophages, and endothelial cells. Quantitation of the neutrophil influx into the bronchial submucosa showed a significant increase compared with uninjured tissue at 24 and 48 hours after injury, P < .05. In conclusion, immunohistochemical detection of activated p65 preceded the overall inflammatory response measured in the lamina propria. However, detection of p65 did not correlate with a recent study showing rapid emigration of neutrophils at 4 hours postinjury. Together, these results suggest that p65 immunostaining may identify cells that are activated to produce proinflammatory cytokines after injury; however, the immunoexpression may not adequately reflect the temporal activation of gene transcription that may occur with proinflammatory cytokine production with inhalation injury.