Fumarate inhibits PTEN to promote tumorigenesis and therapeutic resistance of type2 papillary renal cell carcinoma.

Fumarate is an oncometabolite. However, the mechanism underlying fumarate-exerted tumorigenesis remains unclear. Here, utilizing human type2 papillary renal cell carcinoma (PRCC2) as a model, we show that fumarate accumulates in cells deficient in fumarate hydratase (FH) and inhibits PTEN to activate PI3K/AKT signaling. Mechanistically, fumarate directly reacts with PTEN at cysteine 211 (C211) to form S-(2-succino)-cysteine. Succinated C211 occludes tethering of PTEN with the cellular membrane, thereby diminishing its inhibitory effect on the PI3K/AKT pathway. Functionally, re-expressing wild-type FH or PTEN C211S phenocopies an AKT inhibitor in suppressing tumor growth and sensitizing PRCC2 to sunitinib. Analysis of clinical specimens indicates that PTEN C211 succination levels are positively correlated with AKT activation in PRCC2. Collectively, these findings elucidate a non-metabolic, oncogenic role of fumarate in PRCC2 via direct post-translational modification of PTEN and further reveal potential stratification strategies for patients with FH loss by combinatorial AKTi and sunitinib therapy.

Current and Future Role of Tyrosine Kinases Inhibition in Thyroid Cancer: From Biology to Therapy.

Thyroid cancer represents a heterogenous disease whose incidence has increased in the last decades. Although three main different subtypes have been described, molecular characterization is progressively being included in the diagnostic and therapeutic algorithm of these patients. In fact, thyroid cancer is a landmark in the oncological approach to solid tumors as it harbors key genetic alterations driving tumor progression that have been demonstrated to be potential actionable targets. Within this promising and rapid changing scenario, current efforts are directed to improve tumor characterization for an accurate guidance in the therapeutic management. In this sense, it is strongly recommended to perform tissue genotyping to patients that are going to be considered for systemic therapy in order to select the adequate treatment, according to recent clinical trials data. Overall, the aim of this article is to provide a comprehensive review on the molecular biology of thyroid cancer focusing on the key role of tyrosine kinases. Additionally, from a clinical point of view, we provide a thorough perspective, current and future, in the treatment landscape of this tumor.

Overexpression of G Protein-Coupled Receptor Kinase 6 (GRK6) Is Associated with Progression and Poor Prognosis of Papillary Thyroid Carcinoma.

BACKGROUND Approximately 20% of patients with papillary thyroid carcinoma (PTC) will develop cancer recurrence, but no clinically available biomarker has been identified. Our study aimed to evaluate the prognostic value of G protein-coupled receptor kinase 6 (GRK6) in PTCs. MATERIAL AND METHODS We retrospectively enrolled 108 PTC patients in this study, and explored the expression of GRK6 in resected tumor samples by RT-qPCR and immunohistochemistry (IHC). The clinical data were interpreted by chi-square test, univariate analysis, and multivariate analysis. To investigate the functional mechanisms of GRK6 in regulating PTC progression, we also performed overexpression and silencing experiments in TPC-1 cells, a cell line generated from PTC tissues. RESULTS RT-qPCR results showed a higher level of GRK6-mRNA in PTCs than in adjacent thyroid tissues. IHC revealed a distinct protein expression pattern of GRK6 among PTCs. Accordingly, we classified patients into low-GRK6 and high-GRK6 groups. The chi-square test showed that a higher GRK6 was associated with larger tumor size (P=0.045) and advanced TNM stage (P=0.001). Kaplan-Meier survival curve and log rank test demonstrated that higher GRK6 predicted poor disease-free survival (DFS) in PTC patients (P=0.002). Furthermore, Cox regression analysis confirmed that GRK6 was an independent prognostic factor for a higher recurrence risk of PTCs (P=0.047). MTT assay and Transwell assay demonstrated that GRK6 overexpression can significantly enhance tumor proliferation and invasion, which was consistent with clinical findings. CONCLUSIONS Our data show the oncogenic effects of GRK6 in promoting PTC progression.

Diagnostic value of neutrophil gelatinase-associated lipocalin/matrix metalloproteinase-9 pathway in transitional cell carcinoma of the bladder.

Neutrophil gelatinase-associated lipocalin (NGAL), matrix metalloproteinase (MMP)-9, and NGAL/MMP-9 complex have been evaluated as diagnostic markers of several cancers, but results for bladder cancer are scanty. We evaluated these proteins in urine and serum of 89 patients with histologically confirmed bladder cancer and 119 cancer-free controls from a case-control study. Urinary concentrations were standardized on creatinine level. The performance of these proteins as cancer biomarkers was evaluated through the receiver operating characteristic (ROC) analysis. Urinary level of NGAL, MMP-9, and NGAL/MMP-9 complex was higher in current smokers, whereas no impact of dietary habits was observed. After adjusting for tobacco smoking, urinary concentration of MMP-9 was independently associated with cancer invasiveness, grading, and histological subtype, with elevated concentrations among T2-T4 and non-papillary bladder cancers. Conversely, NGAL and NGAL/MMP-9 complex were significantly higher in non-papillary than in papillary subtype. The pattern was less clear in serum, but correlation between urinary and serum concentration was poor, especially for Ta/is-T1 tumors. The ROC analysis confirmed that MMP-9 was the best marker (area under the ROC curve (AUC) = 0.68). Performances were much greater for muscle-invasive bladder cancers (AUC = 0.90), with elevated negative predictive values (97 %). The present study suggests that NGAL/MMP-9 pathway is associated with an aggressive phenotype of bladder cancer. The elevated negative predictive value of MMP-9 and NGAL/MMP-9 complex makes them candidate markers of exclusion test for bladder cancer. These proteins may be integrated in the surveillance of bladder cancer, thus diminishing patients' discomfort and improving compliance.

miR-29a suppresses growth and metastasis in papillary thyroid carcinoma by targeting AKT3.

MicroRNA-29a (miR-29a) has been reported to play important roles in tumor initiation, development, and metastasis in various cancers. However, the biological function and potential mechanisms of miR-29a in papillary thyroid carcinoma (PTC) remain unclear. In the present study, we discovered that miR-29a was frequently downregulated in PTC tissues, and its expression was significantly associated with tumor size, TNM stage, and lymph node metastasis. Functional assays showed that overexpression of miR-29a markedly suppressed PTC cell proliferation, migration, and invasion and promoted PTC apoptosis and cell cycle arrest at G0/G1 phase. In vivo, miR-29a overexpression decreased tumor growth in a xenograft mouse model. Luciferase reporter assay showed that miR-29a can directly bind to the 3' untranslated region (UTR) of AKT3 in PTC cells. Overexpreesion of miR­29a obviously decreased AKT3 expression, thereby suppressing phosphatidylinositol 3-kinase (PI3K)/AKT pathway activation. We also confirmed that AKT3 expression was increased in PTC tissue and was inversely correlated miR-29a expression in PTC tissues. In addition, downregulation of AKT3 by siRNA mimicked the effects of miR-29a overexpression, and upregulation of AKT3 partially reversed the inhibitory effects of miR-29a. These results suggested that miR-29a could act as a tumor suppressor in PTC by targeting AKT3 and that miR-29a may potentially serve as an anti-tumor agent in the treatment of PTC.

Paraoxonase and arylesterase activities in patients with papillary thyroid cancer.

OBJECTIVE: The aim of this study was to evaluate paraoxonase-1 (PON1) activities and oxidative stress status, and the changes in their levels after total thyroidectomy in patients with papillary thyroid cancer (PTC). MATERIALS AND METHODS: Twenty-five patients with PTC and 27 healthy controls were enrolled in the study. Blood samples were obtained from the PTC patients before and 3 months after the operation. Preoperative and postoperative serum samples from PTC patients and healthy controls were analyzed for paraoxonase (PON), arylesterase (ARE) activities, and lipid hydroperoxide (LOOH) and -SH (total free sulfhydryl) levels. RESULTS: The preoperative PON, ARE and -SH levels of the patients with PTC were significantly lower compared to those of the control group (p = 0.033, p < 0.001, p = 0.002, respectively), while LOOH levels were significantly higher (p < 0.001). The levels of PON and ARE decreased significantly in patients with PTC after the operation (p = 0.038, p = 0.023, respectively), while LOOH and -SH levels remained unchanged (p = 0.117, p = 0.487, respectively). PON and ARE levels showed a positive correlation with -SH (r = 0.211, p = 0.065; r = 0.471, p < 0.001, respectively) and a negative correlation with LOOH (r = - 0.391, p < 0.001, r = - 0.486, p < 0.001, respectively). CONCLUSION: Serum PON1 activity is decreased in patients with PTC, and serum PON1 is positively correlated with -SH, a well-known antioxidant, and negatively correlated with LOOH, an oxidant. PON1 activity is significantly decreased after total thyroidectomy.

Increased CYP24A1 expression is associated with BRAF(V600E) mutation and advanced stages in papillary thyroid carcinoma.

OBJECTIVE: 1α, 25(OH)2 D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1α, 25(OH)2 D3. This study investigates the expression of CYP24A1 and the effect of BRAF(V600E) on its expression in thyroid cancer. METHODS: We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAF(V600E) mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAF(V600E) and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. RESULTS: CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAF(V600E) mutation (P < 0·01). In thyroid cancer cell lines expressing BRAF(V600E) , CYP24A1 expression was significantly higher when compared to those without BRAF(V600E) expression. BRAF(V600E) transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAF(V600E) inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. CONCLUSION: CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAF(V600E) mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAF(V600E) inhibitor PLX4720.

Helicase-like transcription factor: a new marker of well-differentiated thyroid cancers.

BACKGROUND: The preoperative characterization of thyroid nodules is a challenge for the clinicians. Fine-needle aspiration (FNA) is the commonly used pre-operative technique for diagnosis of malignant thyroid tumor. However, many benign lesions, with indeterminate diagnosis following FNA, are referred to surgery. There is an urgent need to identify biomarkers that could be used with the FNA to distinguish benign thyroid nodules from malignant tumors. The purpose of the study is to examine the level of expression of the helicase-like transcription factor (HLTF) in relation to neoplastic progression of thyroid carcinomas. METHODS: The presence of HLTF was investigated using quantitative and semi-quantitative immunohistochemistry in a series of 149 thyroid lesion specimens. Our first clinical series was composed of 80 patients, including 20 patients presenting thyroid adenoma, 40 patients presenting thyroid papillary carcinoma, 12 patients presenting thyroid follicular carcinoma and 8 patients presenting anaplastic carcinoma. These specimens were assessed quantitatively using computer assisted microscopy. Our initial results were validated on a second clinical series composed of 40 benign thyroid lesions and 29 malignant thyroid lesions using a semi-quantitative approach. Finally, the HLTF protein expression was investigated by Western blotting in four thyroid cancer cell lines. RESULTS: The decrease of HLTF staining was statistically significant during thyroid tumor progression in terms of both the percentage of mean optical density (MOD), which corresponds to the mean staining intensity (Kruskall-Wallis: p < 0.0005), and the labelling index (LI), which corresponds to the percentage of immunopositive cells (Kruskall-Wallis: p < 10-6). Adenomas presented very pronounced nuclear HLTF immunostaining, whereas papillary carcinomas exhibited HLTF only in the cytoplasm. The number of HLTF positive nuclei was clearly higher in the adenomas group (30%) than in the papillary carcinomas group (5%).The 115-kDa full size HLTF protein was immunodetected in four studied thyroid cancer cell lines. Moreover, three truncated HLTF forms (95-kDa, 80-kDa and 70-kDa) were also found in these tumor cells. CONCLUSIONS: This study reveals an association between HLTF expression level and thyroid neoplastic progression. Nuclear HLTF immunostaining could be used with FNA in an attempt to better distinguish benign thyroid nodules from malignant tumors.

HER2/neu gene amplification determines the sensitivity of uterine serous carcinoma cell lines to AZD8055, a novel dual mTORC1/2 inhibitor.

OBJECTIVE: To evaluate c-erbB2 gene amplification in a series of primary uterine serous carcinoma (USC) cell lines. To assess the efficacy of AZD8055, a novel dual mTORC1/2 inhibitor against primary HER2/neu amplified vs HER2/neu not amplified USC cell lines. METHODS: Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by FISH assays. In vitro sensitivity to AZD8055 was evaluated by flow-cytometry-based viability and proliferation assays. Cell cycle profile and downstream cellular responses to AZD8055 were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. RESULTS: Nine of 22 (40.9%) USC cell lines demonstrated c-erbB2 gene amplification by FISH. AZD8055 caused a strong differential growth inhibition in USC cell lines, with high HER-2/neu-expressors demonstrating significantly higher sensitivity when compared to low HER-2/neu-expressors (AZD-8055 IC50 mean±SEM=0.27±0.05µM in c-erbB2 amplified versus 1.67±0.68µM in c-erbB2 not amplified tumors, P=0.03). AZD8055 growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G0/G1 cell cycle phase and a dose-dependent decline in pS6 levels in both c-erbB2 amplified vs c-erbB2 not amplified USC cell lines. CONCLUSIONS: AZD8055 may represent a novel targeted therapeutic agent in patients harboring advanced/recurrent/refractory USC. c-erbB2 gene amplification may represent a biomarker to identify USC patients who may benefit most from the use of AZD8055.

Prognostic significance and diagnostic value of PTEN and p53 expression in endometrial carcinoma. A retrospective clinicopathological and immunohistochemical study.

PURPOSE: To investigate the PTEN and p53 gene expression in endometrioid and serous papillary endometrial carcinomas and clarify their prognostic significance by studying the PTEN and p53 expression in relation to tumor stage and grade. METHODS: Archival pathological sections of 61 cases with endometrial cancer examined in a 5-year-period (January 2006-December 2010) were retrieved and re-examined. Immunohistochemical investigation was performed by the Ventana system. Anti-PTEN and anti-p53 monoclonal antibodies were used. Disease staging was made according to the FIGO staging system. RESULTS: Forty-nine (80.32%) cases were endometrioid adenocarcinomas. Patient age ranged from 39-75 years (mean 62.5). Grade 1 tumors:19/22 (86.3%) cases had stage Ib, 2/22 (9.09%) stage Ic and 1/22 (4.54%) stage IIIc. Eighteen of 22 (81.8%) cases were PTEN positive and 4/22 (18.2%) p53 positive. Grade 2 tumors: 17/ 23 (73.91%) cases had stage I b, 4/23 (17.39%) stage Ic and 2/23 (8.69%) stage IIIc. Seventeen of 23 (73.91%) cases were PTEN positive and 47sol;23 (17.3%) p53 positive. Grade 3 tumors: 2/4 (50%) cases had stage Ic and 2/4 (50%) stage IIIc. No case was PTEN positive and 2/4 (50%) were p53 positive. Twelve (19.35%) cases were serous papillary carcinomas. Patient age ranged from 63-79 years (mean 76). Five (41.66%) cases had stage Ic and 5 (41.66%) stage IIIc, with nodal metastases and peritoneal involvement. Two (16.66%) cases developed on endometrial polyps with minimal myometrial involvement (stage Ib) and in both cases elements of endometrioid adenocarcinoma were observed as well. Immunohistochemical study showed that 11 (91.66%) cases were p53 positive and 2 (16.66%) PTEN positive. CONCLUSION: PTEN and p53 immunoexpression helps both in accurate diagnosis and proper therapeutic approach of the various endometrial carcinomas. PTEN and p53 are also prognostic markers for these kind of tumors.

Thyroid Follicular Cell-derived Carcinomas in a Background of Multiple Adenomatous Nodules Leading to a Diagnosis of PTEN Hamartoma Tumor Syndrome in an Adult Patient With a Novel RECQL4 Mutation.

BACKGROUND: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is a complex disorder. Carriers develop hamartomatous tumors, with an increased risk for developing malignant tumors in multiple organs. Surveillance to facilitate the early detection and treatment of malignancies is extremely important. CASE REPORT: A 31-year-old male presented with a 10 cm left lobe thyroid gland mass. After fine needle aspiration a left hemithyroidectomy was performed, which demonstrated a minimally invasive follicular thyroid carcinoma (FTC, stage pT3a) and microscopic classical papillary thyroid carcinoma (PTC) in the background of about 50 separate adenomatous nodules (0.2-5 mm). Immunostaining showed loss of PTEN protein in the minimally invasive FTC and in all of the nodules tested, with uninvolved parenchyma serving as an internal control. Kaiser Permanente Northern California (KPNC) Hereditary Cancer Panel, testing for 62 genes, was performed and showed germline mutations in PTEN and RecQ like helicase 4 (RECQL4) genes. Completion thyroidectomy subsequently performed demonstrated about 60 follicular cell-derived adenomatous nodules (0.3-10 mm). Genetic counseling and evaluation documented Cowden syndrome (CS) in the family. Thus, PHTS was confirmed. CONCLUSION: This report documents synchronous FTC and PTC in a background of multiple follicular adenomatous nodules with a novel RECQL4 mutation in an adult patient with PHTS. As such, documented the loss of PTEN protein in a thyroid gland affected by multiple adenomatous nodules aided in diagnosing PHTS.

Insulin-like growth factor-I receptor inhibition by specific tyrosine kinase inhibitor NVP-AEW541 in endometrioid and serous papillary endometrial cancer cell lines.

PURPOSE: The role of the insulin-like growth factor (IGF) system in endometrial cancer has been well established. The IGF-I receptor (IGF-IR) emerged as a promising therapeutic target in a number of cancers. NVP-AEW541 (Novartis Pharma) is a pyrrolo(2,3-d)pyrimidine derivative with specific IGF-IR tyrosine kinase inhibitory activity. NVP-AEW541 has been shown to abrogate IGF-I-mediated IGF-IR autophosphorylation and to reduce activation of the IGF-IR signaling pathways. The aim of the present study was to investigate the anti-proliferative activity of NVP-AEW541 in Type I (endometrioid) and Type II (uterine serous papillary endometrial carcinoma, USPC) endometrial cancer cell lines. METHODS: Type I (ECC-1, Ishikawa) and Type II (USPC-1, USPC-2) endometrial cancer cell lines were treated with NVP-AEW541 in the presence of IGF-I, and the following parameters were measured: IGF-IR, AKT and ERK phosphorylation, apoptosis, proliferation, cell cycle progression and IGF-IR internalization. RESULTS: Results obtained showed that NVP-AEW541 abolished the IGF-I stimulated IGF-IR phosphorylation in all of the cell lines investigated, whereas it abolished AKT and ERK phosphorylation preferentially in ECC-1 and USPC-1 cells. Furthermore, the inhibitor prevented from IGF-I from exerting its antiapoptotic effect in ECC-1, USPC-1 and USPC-2 cells. In addition, proliferation assays showed that NVP-AEW541 caused a decrease in proliferation rate in all of the cell lines. NVP-AEW541 had no major effect on the insulin receptor. CONCLUSION: Our results suggest that specific IGF-IR inhibition by NVP-AEW541 might be a promising therapeutic tool in endometrial cancer.

Oncoprotein signaling mediates tumor-specific inflammation and enhances tumor progression.

The RET/PTC3 (RP3) fusion protein is an oncogene expressed during the development of thyroid cancer and in thyroid epithelial cells of patients with Hashimoto's thyroiditis. RP3 has two immunological properties: 1) it encodes a chimeric protein including peptides that may be targets of antitumor immune responses and 2) it is a tyrosine kinase that can activate NF-kappaB transcriptional programs, induce secretion of proinflammatory mediators, and stimulate innate immunity. To distinguish the antigenic properties of the RP3 oncoprotein from its signaling function, a transplantable tumor system was developed. Tumors expressing the functional, but not mutant, form of RP3 show enhanced infiltration of CD8(+) lymphocytes, myeloid-derived CD11b(+)Gr1(+) cells, and enhanced growth in immunocompetent mice. In contrast, RP3 signaling mutant-expressing tumors maintained enhanced infiltration of CD8(+) lymphocytes did not enhance recruitment of CD11b(+)Gr1(+) cells and showed a decreased tumor incidence. These results implicate a role for RP3 function in enhancing a tumor-suppressive innate inflammatory response. These experiments support a mechanism whereby oncogenes can directly recruit and activate innate and adaptive immune cells, resulting in enhanced tumor progression.

Inhibition of FGF10-ERK signal activation suppresses intraductal papillary neoplasm of the bile duct and its associated carcinomas.

Evidence regarding intraductal papillary neoplasm of the bile duct (IPNB) as a type of precancerous lesion of cholangiocarcinoma is limited. Moreover, a reproducible in vivo model is lacking, and IPNB pathogenesis remains unclear. Here, we use a doxycycline-inducible tetracycline (Tet)-on mice model to control fibroblast growth factor 10 (FGF10) expression, which regulates branching and tubule formation. FGF10-induced IPNB mimics the multifocal and divergent human IPNB phenotypes via the FGF10-FGF receptor 2 (FGFR2)-RAS-extracellular-signal-regulated kinase (ERK) signaling pathway. A paracrine/autocrine growth factor is sufficient to initiate and maintain IPNB originating from the peribiliary glands, including biliary stem/progenitor cells. With KrasG12D, p53, or p16 mutations or both, Fgf10-induced IPNB shows stepwise carcinogenesis, causing associated invasive carcinoma. Fgf10-induced papillary changes and progression are suppressed by the inhibition of the FGF10-FGFR2-RAS-ERK signaling pathway, demonstrating that the signal is a therapeutic target for IPNB and associated carcinoma.

Depletion of the human Nα-terminal acetyltransferase A induces p53-dependent apoptosis and p53-independent growth inhibition.

The human protein N(α)-terminal acetyltransferase A complex (hNatA), composed of the catalytic hNaa10p (hArd1) and auxiliary hNaa15p (hNat1/NATH/Tubedown) subunits, was reported to be important for cell survival and growth of various types of cancer. However, little is known about the mechanisms mediating growth inhibition and apoptosis following loss of hNatA function. Here, we have screened 11 different thyroid cell lines for hNAA10 RNAi phenotypes and observed mostly growth inhibition, which was independent of TP53 functional status and developed by several different mechanisms involving (i) downregulation of cyclin D1, (ii) increase in p27/Kip1 and (iii) inactivation of Rb/E2F pathway. hNatA depletion in aggressive thyroid cancer cell lines (8305C, CAL-62 and FTC-133) with mutated TP53 increased sensitivity to drug-induced cytotoxicity, but in a cell type specific manner: 8305C (TRAIL), CAL-62 (daunorubicin) and FTC-133 (troglitazone). Cells harboring wild-type TP53 were also prone to apoptosis via the p53 pathway after hNatA downregulation. Importantly, in hNatA-depleted cells DNA-damage signaling was activated in the absence of exogenous DNA damage independent on TP53 status. Our findings indicate that several mechanisms of growth inhibition and apoptosis may be induced by hNatA knockdown and that hNatA knockdown could be exploited for use in combinatorial chemotherapy.

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.

Higher sensitivity to patupilone versus paclitaxel chemotherapy in primary uterine serous papillary carcinoma cell lines with high versus low HER-2/neu expression in vitro.

OBJECTIVE: To compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine serous papillary carcinoma (USPC) with high versus low HER-2/neu expression. METHODS: Six primary USPC cell lines, half of which overexpress HER-2/neu at a 3+ level, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. Quantitative RT-PCR was used to identify potential mechanisms underlying the differential sensitivity/resistance to patupilone versus paclitaxel in primary USPC cell lines. RESULTS: Cell lines overexpressing HER-2/neu showed higher proliferation when compared to low HER-2/neu-expressing cell lines. Compared to low-expressing cell lines, high HER-2/neu expressors were significantly more sensitive to patupilone than to paclitaxel (P<0.0002). In contrast, there was no appreciable difference in sensitivity to patupilone versus paclitaxel in primary USPC cell lines with low HER-2/neu expression. Higher levels of ß-tubulin III (TUBB3) and P-glycoprotein (ABCB1) were detected in USPC cell lines with high versus low HER-2/neu expression (P<0.05). CONCLUSIONS: USPC overexpressing HER-2/neu display greater in vitro sensitivity to patupilone and higher levels of the patupilone molecular target TUBB3 when compared to low HER-2/neu expressors. Due to the adverse prognosis associated with HER-2/neu overexpression in USPC patients, patupilone may represent a promising novel drug to combine to platinum compounds in this subset of aggressive endometrial tumors.

COX-2 is induced by HGF stimulation in Met-positive thyroid papillary carcinoma cells and is involved in tumour invasiveness.

Thyroid papillary carcinoma (TPC) cells express high levels of cytoplasmic cyclo-oxygenase 2 protein. Analysis of microdissected samples of the tumour and of the paired normal thyroid tissue confirmed that mRNA transcripts for cyclo-oxygenase 2 (COX-2) were significantly more numerous in the tumour (7.6 +/- 13-fold; p = 0.01). High levels of COX-2 mRNA were not associated with age, sex, tumour size or lymph node metastasis. COX-2 was not homogeneously expressed throughout the tumour, but was significantly higher at the tumour invasion front. Hepatocyte growth factor (HGF) can up-regulate the expression of COX-2 mRNA. A marked increase in COX-2 mRNA levels was observed in 8/8 primary TPC cultures after HGF stimulation (6.3 +/- 6-fold) and in two papillary carcinoma cell lines (TPC-1 and NPA). Specific involvement of the high-affinity HGF receptor (Met protein) was suggested by the observation that PHA-665752, an inhibitor of the catalytic activity of c-Met kinase, caused a 54% reduction of the hepatocyte growth factor-induced COX-2 up-regulation. The possibility that HGF-Met interactions also had a causative role in the up-regulation of COX-2 in vivo was investigated in 30 tumour samples, where it was found that there was a statistically significant correlation (p = 0.001, r = 0.85) in the levels of expression of MET and COX-2 RNAs. The biological role of COX-2 in TPC cells was investigated by treating the TPC cell lines with the specific COX-2 inhibitor NS-398. It was found that NS-398 treatment significantly reduced the migration (50-75%) and invasiveness (47-92%) of tumour cells, but did not alter cell proliferation. Our data suggest that the increased expression of Met protein in TPC cells has a role in up-regulating the expression of COX-2, which in turn contributes to the invasive capacity of TPC cells.

Notch1 mediates growth suppression of papillary and follicular thyroid cancer cells by histone deacetylase inhibitors.

Notch1 is a multifunctional transmembrane receptor that regulates cellular differentiation, development, proliferation, and survival in a variety of contexts. We have previously shown that Notch1 may function as a tumor suppressor and that histone deacetylase (HDAC) inhibitors can induce Notch1 expression in some endocrine cancers. Here, we showed that although there was minimal Notch1 expression in follicular thyroid cancer FTC236 and papillary thyroid cancer DRO cells, transfection of constitutive Notch1 plasmid into these cells led to growth inhibition, down-regulation of cyclin D1, and up-regulation of p21. Treatment of FTC236 cells with HDAC inhibitors valproic acid (1-4 mmol/L) or suberoyl bishydroxamic acid (10-30 micromol/L) induced functional Notch1 protein expression and suppressed cell growth in a dose-dependent manner. Notch1 siRNA interference blocked the antiproliferative effect of HDAC inhibitors. Western blot analysis revealed the reduction of cyclin D1 and the increase of p21 in HDAC inhibitor-treated cells. These results indicate that HDAC inhibitors activate Notch1 signaling in thyroid cancer cells and lead to the suppression of proliferation by cell cycle arrest. Our findings provide the first documentation of the role of Notch1 signaling as a tumor suppressor in DRO and FTC236 cells, suggesting that Notch1 activation may be a potential therapeutic target for papillary and follicular thyroid cancers.

Significance of DNA methyltransferase-1 and histone deacetylase-1 in pancreatic cancer.

Epigenetic modifications play an important role during carcinogenesis. The main goal of this study was to examine expression levels of two critical enzymes, DNA methyltransferase-1 (DNMT1) and histone deacetylase-1 (HDAC1), by immunohistochemistry (IHC) in human pancreatic cancer and precancerous lesions: 20 foci containing normal ductal epithelial cells without an inflammatory back-ground (DE), 30 containing ductal epithelial cells with an inflammatory background (DEI), 48 of pancreatic intraepithelial neoplasia-1A (PanIN-1A), 103 of PanIN-1B, 99 of PanIN-2, 30 of PanIN-3, 18 of intraductal papillary mucinous neoplasm A (IPMA), 10 of IPMB, 20 of IPMC, and 54 of pancreatic ductal adenocarcinoma (PDAC). The expression levels of both DNMT1 and HDAC1 increased from normal to precancerous lesions to pancreatic cancer, in a malignancy-dependent manner. Correlations between expression levels and clinicopathological features of the 54 PDAC patients were also analyzed. The expression of DNMT1 significantly correlated with nerve infiltration, degree of tumor differentiation and TNM staging (p<0.05), while that of HDAC1 correlated with proliferative activity, degree of tumor differentiation and TNM staging (p<0.05). Patients with higher expression of DNMT1 and/or HDAC1 had an overall lower survival than those with lower expression (p<0.05). Higher expression of DNMT1 and HDAC1 correlated with advanced stages of the disease and reflect the malignancy of pancreatic carcinoma. They may become new prognostic markers and potential therapeutic targets for pancreatic cancer.