Edgard Graner

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16‐F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16‐F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16‐F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16‐F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.

Myofibroblasts in the stroma of oral squamous cell carcinoma are associated with poor prognosis

with lupus panniculitis. The core biopsy specimen, taken from the recurrent mass in 2003, demonstrated lymphoplasmacytic infiltrates similar to those seen in the previous excision specimen. Surgical intervention was deemed unnecessary and a conservative approach was employed. Lupus mastitis is a rare presentation of lupus profundus, usually following the clinical manifestations of SLE or chronic discoid lupus erythematosus (DLE). In some cases involvement of the fat precedes SLE for months. To our knowledge, only about 16 cases of lupus mastitis have been reported, occurring mainly in association with chronic DLE. The typical presentation of the elementary lesion is a deep subcutaneous nodule or swelling. The overlying skin can be normal or show typical features of discoid lupus. In the breast, fat necrosis commonly mimics malignancy clinically. A history of trauma is absent in most cases at this site. However, fat necrosis caused by trauma is common in the breast and differs from lupus profundus, as the latter process is centred around blood vessels and connective tissue, associated with heavy perivascular and periadnexal lymphocytic infiltrates and hyaline sclerosis of the dermal collagen. It is this feature which is responsible for the hard ‘carcinoma’like feel to lupus mastitis and its dense opacity on mammography. Fat necrosis in the breast is seen in other connective tissue disorders and relevant clinical information is essential when lupus profundus presents at this site. Our case illustrates the importance of providing relevant clinical information to histopathologists to make a diagnosis of lupus mastitis. Lupus mastitis should be suspected in a patient with SLE presenting with a recurrent painful mass in the breast. Equally, acknowledging the increased incidence of breast carcinoma in SLE patients and thus obtaining biopsy confirmation is essential. Lymphocytic vasculitis, sclerosis and hyaline panniculitis in the biopsy specimen would confirm the diagnosis of lupus mastitis and avoid unnecessary excisions which, in these patients, have been reported to lead to atrophic scarring or chronic ulcers. E Nigar K Contractor H Singhal R N Matin Departments of Pathology and Surgery and Dermatology, Northwick Park Hospital, North West London Hospitals NHS Trust, Harrow, UK 1. Irgang S. Lupus erythematosus profundus: report of an example with clinical resemblance to Darier-Roussy sarcoid. Arch. Derm. Syphilol. 1940; 42; 97–108. 2. Holland NW, McKnight K, Challa VR, Agudelo CA. Lupus panniculitis (profundus) involving the breast: report of two cases and review of the literature. J. Rheumatol. 1995; 22; 344–346. 3. Georgian-Smith D, Lawton TJ, Moe RE, Couser WG. Lupus mastitis: radiologic and pathologic features. AJR Am. J. Roentgenol. 2002; 178; 1233–1235. 4. Cernea SS, Kihara M, Sotto MN, Vilela Ma. Lupus mastitis. J. Am. Acad. Dermatol. 1993; 29; 343–346. 5. De Bandt M, Meyer O, Grossin M, Khan MF. Lupus mastitis heralding systemic lupus erythematosus with antiphospholipid syndrome. J. Rheumatol. 1993; 20; 1217–1220. 6. Ujiie H, Shimizu T, Ito M, Arita K, Shimizu H. Lupus erythematosus profundus successfully treated with dapsone: review of the literature. Arch. Dermatol. 2006; 142; 399–401. 7. Cerveira I, Costa Matos L, Garrido A et al. Lupus mastitis. Breast 2006; 15; 670–672. 8. Bernatsky S, Ramsey-Goldman R, Boivin JF et al. Do traditional Gail model risk factors account for increased breast cancer in women with lupus? J. Rheumatol. 2003; 30; 1505–1507.

Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of activin A

Myofibroblasts are essential during wound healing and are often found in the stroma of oral squamous cell carcinomas (OSCC). Although the molecular mechanisms by which myofibroblasts influence OSCC remain largely unknown, previous studies demonstrated that presence of myofibroblast in OSCC stroma is an important risk factor of patients shortened survival. Here we showed that some growth factors are produced in higher levels by tumor-associated myofibroblasts compared to tumor-associated fibroblasts, including activin A. Myofibroblast-conditioned media containing activin A significantly increased OSCC cell proliferation and tumor volume, whereas down-regulation of activin A in the conditioned media decreased proliferation. In addition, myofibroblasts induced in vitro invasion of OSCC cells, which was accompanied by an increased production of matrix metalloproteinases (MMP). In vivo, a significant correlation between presence of myofibroblasts and activities of MMP-2 and MMP-9 was observed in OSCC samples. However, blockage of activin A synthesis by myofibroblasts did not affect invasion and MMP production by OSCC cells. Together, our data demonstrate that activin A is required for the proliferative effects of myofibroblasts on OSCC cells. We conclude that myofibroblasts in the stroma of OSCC may influence proliferation and invasion, resulting in more aggressive tumor.

The Fatty Acid Synthase Inhibitor Orlistat Reduces the Growth and Metastasis of Orthotopic Tongue Oral Squamous Cell Carcinomas

Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC. Mol Cancer Ther; 13(3); 585–95. ©2013 AACR.

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

Oral paracoccidioidomycosis: a study of 36 South American patients

Paracoccidioidomycosis (South American blastomycosis) is an uncommon, progressive systemic mycosis, virtually only seen in persons who have visited Latin America. Reports of oral lesions are extremely rare in the English-language literature. Thirty-six adults with oral lesions as the first sign of paracoccidioidomycosis are described; this appears to be the largest series in the dental literature. All had chronic proliferative mulberry-like ulcerated oral lesions; the diagnosis was confirmed histologically. The gingiva or alveolar process was the typical site, but lesions were also seen particularly on the palate and lip. Most of the patients proved to have detectable pulmonary involvement. Patients with lesions in the oropharynx, tongue, or floor of mouth all had confirmed pulmonary lesions.

HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis

BackgroundHOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer.MethodsIn order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression.ResultsWe showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026).ConclusionOur findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.

Lack of association between IRF6 polymorphisms (rs2235371 and rs642961) and non-syndromic cleft lip and/or palate in a Brazilian population

BACKGROUND Interferon regulatory factor 6 (IRF6) gene has emerged as a potential susceptibility gene for non-syndromic cleft lip and/or palate (NSCL/P) in different populations. The aim of this study was to determine the association of IRF6 rs2235371 and rs642961 polymorphisms with NSCL/P in a Brazilian population. METHODS Two hundred and twenty-eight patients affected by NSCL/P and 126 healthy individuals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. RESULTS Overall genotype distributions of rs2235371 and rs642961 polymorphisms were as expected by Hardy-Weinberg equilibrium test. The rs2235371 polymorphic genotype GA was identified in 10.1% of the patients with NSCL/P and in 10.3% of the control group, revealing no statistical difference. Similarly, the frequency of rs642961 minor genotypes (GA and AA) was quite similar between control group (28.6%) and NSCL/P group (25.4%), without significant difference. CONCLUSION Our findings are consistent with a lack of involvement of IRF6 rs2235371 and rs642961 polymorphisms in the NSCL/P pathogenesis in the Brazilian population.

Clinicopathological prognostic factors of oral tongue squamous cell carcinoma: A retrospective study of 202 cases

Although several histopathological parameters and grading systems have been described as predictive of the treatment response and outcome of oral squamous cell carcinoma (OSCC), none is universally accepted. A new scoring system, the histological risk model, was recently described to be a powerful predictive tool for recurrence and overall survival in OSCC. The aim of this study was to verify the predictive role of the histological risk model in a cohort of 202 patients at all stages of oral/mobile tongue squamous cell carcinoma (OTSCC). Demographic and clinical data were collected from the medical records and the tumours were evaluated using the histological risk model. Statistical analyses were performed using the χ(2) test, the Kaplan-Meier method, and the Cox regression model. The histological risk model showed no statistical correlation with demographic or clinical parameters and did not Predict the outcome of the OTSCC patients. However, multivariate regression analysis revealed a significant correlation of the clinical disease stage with the disease outcome. Despite major efforts to identify new predictive parameters and histological systems, clinical features are still the most reliable prognostic factors for patients with OTSCC.

Clinicopathological significance of ubiquitin-specific protease 2a (USP2a), fatty acid synthase (FASN), and ErbB2 expression in oral squamous cell carcinomas

Overexpression of fatty acid synthase (FASN) and ErbB2 has been described in oral squamous cell carcinomas (OSCC). FASN is the key lipogenic enzyme responsible for the endogenous synthesis of fatty acids and its expression can be regulated by ErbB2. The deubiquitinating enzyme (DUB) ubiquitin-specific protease 2a (USP2a) plays a critical role in prostate cancer cell survival by stabilizing the FASN protein. This study investigates whether the gene expression and the immunohistochemical status of FASN, ErbB2, and USP2a correlate with the clinicopathological characteristics of OSCC cases. A strong positive correlation among ErbB2, FASN, and USP2a expression (p=0.001) was observed by qRT-PCR in laser capture microdissected OSCC samples. Perineural infiltration was associated with ErbB2 mRNA expression (p=0.046). The presence of metastatic cervical lymph nodes was associated with FASN (p=0.002), ErbB2 (p=0.001), and USP2a (p=0.006) mRNA levels. ErbB2 staining at the cell membranes was stronger in well-differentiated lesions while a cytoplasmic positivity was found in poorly differentiated tumors. Most of the OSCC (97.06%) that showed a high positivity for FASN were also labeled for ErbB2 at the cell membranes (p=0.001). FASN and ErbB2 positivity was associated with tumor thickness and lymphatic embolization (p=0.006 and p=0.035, p=0.006 and p=0.024 respectively). The membrane expression of ErbB2 as well as FASN and Ki-67 staining were significantly associated with a high risk of recurrence by predicting both disease free survival (log-rank test, p=0.0056, p=0.0011, and p=0.0004, respectively) and overall survival (log-rank test, p=0.0005, p=0.0062, and p=0.0001, respectively). Taken together, the results presented here suggest a molecular connection among FASN, ErbB2, and USP2a in OSCC since their mRNA and protein levels were associated with tumor progression and poor prognosis.