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Background At diagnosis, more than 80% of patients with pancreatic cancer (PC) suffer from significant weight loss due to malnutrition which is a major concern for patient management, and this may negatively impact treatment outcomes and patient prognosis. Patients and Methods We performed an observational, retrospective study on patients with metastatic PC (mPC) undergoing first-line chemotherapy with nab-Paclitaxel containing schedules and receiving or not receiving nutritional support (NS) and pancreatic enzyme replacement therapy (PERT) to investigate their relevance in this setting. Results We observed that PERT and ancillary dietary interventions are related to longer overall survival (OS; median: 16.5 vs. 7.5 months, P < .001) and have a significant, independent, prognostic impact for better outcomes (P = .013), independently from the therapeutic regimen. Furthermore, PERT and NS prevented weight loss during chemotherapy and obtained an improvement of nutritional parameters such as phase angle and free-fat mass index, after 3 months of anticancer treatment. Consistently, the positive impact on OS correlated also with the prevention of Karnofsky performance status deterioration and a lower incidence of maldigestion-related symptoms. Conclusions Our data suggest that an early and well-conducted NS in patients with mPC may impact on survival and preserve performance status, thus improving quality of life.

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Supplementary Figure 4 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Schematic representation of the transcriptional interactions among 6 cell cycle-related genes down-regulated in shTRAP1 cells according to the whole genome gene expression analysis and confirmed by subsequent Real Time validation analysis (query genes, visualized in green) and MAPK pathway (visualized in red). Transcriptional interactions from IPA software.

TRAP1 and BRAF protein levels in 41 human BRAF-wild type colorectal carcinomas expressed as time increase in tumor compared to correspondent non-infiltrated peritumoral mucosas.

A. Gene ontology categories associated to genes up/downregulated upon TRAP1 silencing in HCT116 cells. B. Top predicted biofunctions associated to genes downregulated upon TRAP1 silencing in HCT116 cells. Color intensity for each biofunction corresponds to p-value. Biofunction prediction from Fidea software. C. Real time gene expression analysis of previously validated genes in HT29 cells upon TRAP1 transient interference (*p=

TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca2+-binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway. Cancer Res; 70(16); 6577–86. ©2010 AACR.

The Ca2+-binding protein sorcin regulates intracellular calcium homeostasis and plays a role in the induction of drug resistance in human cancers. Recently, an 18 kDa mitochondrial isoform of sorcin was reported to participate in antiapoptosis in human colorectal cancer (CRC), but information remains lacking about the functional role of the more abundant 22 kDa isoform of sorcin expressed in CRC. We found the 22 kDa isoform to be widely expressed in human CRC cells, whether or not they were drug resistant. Its upregulation in drug-sensitive cells induced resistance to 5-fluorouracil, oxaliplatin, and irinotecan, whereas its downregulation sensitized CRC cells to these chemotherapeutic agents. Sorcin enhances the accumulation of Ca2+ in the endoplasmic reticulum (ER), preventing ER stress, and, in support of this function, we found that the 22 kDa isoform of sorcin was upregulated under conditions of ER stress. In contrast, RNAi-mediated silencing of sorcin activated caspase-3, caspase-12, and GRP78/BiP, triggering apoptosis through the mitochondrial pathway. Our findings establish that CRC cells overexpress sorcin as an adaptive mechanism to prevent ER stress and escape apoptosis triggered by chemotherapeutic agents, prompting its further investigation as a novel molecular target to overcome MDR. Cancer Res; 71(24); 7659–69. ©2011 AACR.

A-B. Total cell lysates from siTRAP1 HCT116 and HT29 cells (A) or HCT116 cells transfected with pMock or TRAP1 cDNA (B) were separated by SDS-PAGE and immunoblotted with indicated antibodies. C. Cell cycle distribution in HCT116 cells transfected individually or in combination with negative, TFDP1, PBK, CCNE1, or NEK6 siRNA (*p=

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HSP990 induces downregulation of specific protein of TRAP1 network and inhibits cell proliferation. A-B. Mitochondrial (A) and total cell (B) lysates from HT29 cells were separated by SDS-PAGE and immunoblotted with anti-sorcin and anti-VDAC (A) or anti-eEF1G, anti-eIF4A, anti-eIF4E and anti-GAPDH (B) antibodies. C-D. Inhibition of cell proliferation (C) and induction of apoptosis (D) in HT29 cells treated with increasing concentrations of AUY922 and HSP990 for indicated time points. C. *p

Supplementary Figure Legends 1- 4 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Supplementary Figure 2 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

TRAP1 silencing results in reduced BRAF-wt or V600E protein levels, attenuation of ERK phosphorylation and cell cycle progression. A-B. Total cell lysates from serum-starved COLO320 cells transfected with negative or TRAP1 siRNA and further serum stimulated for 1 and 3h (A) or from scramble and shTRAP1 MCF7 cells transfected with BRAF-wt or BRAF-V600E constructs were separated by SDS-PAGE and immunoblotted with indicated antibodies. C-D. Cell cycle distribution in MCF7 (C) and COLO320 (D) cells transfected with negative or TRAP1 siRNA. Inserts: Total cell lysates from MCF7 (C) and COLO320 (D) cells transfected with negative or TRAP1 siRNA were separated by SDS-PAGE and immunoblotted with indicated antibodies.

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Supplementary Figure 1 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

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Supplementary Figure 3 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Supplementary Figure Legends and Experimental Procedures

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Supplementary Table 1 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Genes differently expressed in HCT116 TRAP1-silenced cells. Genes were selected with differential score cutoff set at {plus minus}30 (p < 0.001).

Insulin-like growth factor binding protein 6 (IGFBP6) is a secreted protein with a controversial role in human malignancies, being downregulated in most types of human cancer, but upregulated in selected tumors. Ovarian cancer (OC) is a human malignancy characterized by IGFBP6 downregulation; however, the significance of its low expression during ovarian carcinogenesis is still poorly understood. In the present study, IGFBP6 expression and activation of its associated signaling pathway were evaluated in two matched OC cell lines derived from a high-grade serous OC before and after platinum resistance (PEA1 and PEA2 cells, respectively). A whole genome gene expression analysis was comparatively performed in both cell lines upon IGFBP6 stimulation using Illumina technology. IGFBP6 gene expression data from human OC cases were obtained from public datasets. Gene expression data from public datasets confirmed the downregulation of IGFBP6 in primary and metastatic OC tissues compared with in normal ovarian tissues. The comparative analysis of platinum-sensitive (PEA1) and platinum-resistant (PEA2) cell lines showed quantitative and qualitative differences in the activation of IGFBP6 signaling. Notably, IGFBP6 enhanced ERK1/2 phosphorylation only in PEA1 cells, and induced more evident and significant gene expression reprogramming in PEA1 cells compared with in PEA2 cells. Furthermore, the analysis of selected genes modulated by IGFBP6 (i.e.

Metabolic rewiring fuels rapid cancer cell proliferation by promoting adjustments in energetic resources, and increasing glucose uptake and its conversion into lactate, even in the presence of oxygen. Furthermore, solid tumors often contain hypoxic areas and can rapidly adapt to low oxygen conditions by activating hypoxia inducible factor (HIF)-1α and several downstream pathways, thus sustaining cell survival and metabolic reprogramming. Since TNF receptor-associated protein 1 (TRAP1) is a HSP90 molecular chaperone upregulated in several human malignancies and is involved in cancer cell adaptation to unfavorable environments and metabolic reprogramming, in the present study, its role was investigated in the adaptive response to hypoxia in human colorectal cancer (CRC) cells and organoids. In the present study, glucose uptake, lactate production and the expression of key metabolic genes were evaluated in TRAP1-silenced CRC cell models under conditions of hypoxia/normoxia. Whole genome gene expression profiling was performed in TRAP1-silenced HCT116 cells exposed to hypoxia to establish the role of TRAP1 in adaptive responses to oxygen deprivation. The results revealed that TRAP1 was involved in regulating hypoxia-induced HIF-1α stabilization and glycolytic metabolism and that glucose transporter 1 expression, glucose uptake and lactate production were partially impaired in TRAP1-silenced CRC cells under hypoxic conditions. At the transcriptional level, the gene expression reprogramming of cancer cells driven by HIF-1α was partially inhibited in TRAP1-silenced CRC cells and organoids exposed to hypoxia. Moreover, Gene Set Enrichment Analysis of TRAP1-silenced HCT116 cells exposed to hypoxia demonstrated that TRAP1 was involved in the regulation of ribosome biogenesis and this occurred with the inhibition of the mTOR pathway. Therefore, as demonstrated herein, TRAP1 is a key factor in maintaining HIF-1α-induced genetic/metabolic program under hypoxic conditions and may represent a promising target for novel metabolic therapies.

Regulation of tumour cell proliferation by molecular chaperones is still a complex issue. Here, the role of the HSP90 molecular chaperone TRAP1 in cell cycle regulation was investigated in a wide range of human breast, colorectal, and lung carcinoma cell lines, and tumour specimens. TRAP1 modulates the expression and/or the ubiquitination of key cell cycle regulators through a dual mechanism: (i) transcriptional regulation of CDK1, CYCLIN B1, and MAD2, as suggested by gene expression profiling of TRAP1-silenced breast carcinoma cells; and (ii) post-transcriptional quality control of CDK1 and MAD2, being the ubiquitination of these two proteins enhanced upon TRAP1 down-regulation. Mechanistically, TRAP1 quality control on CDK1 is crucial for its regulation of mitotic entry, since TRAP1 interacts with CDK1 and prevents CDK1 ubiquitination in cooperation with the proteasome regulatory particle TBP7, this representing the limiting factor in TRAP1 regulation of the G2-M transition. Indeed, TRAP1 silencing results in enhanced CDK1 ubiquitination, lack of nuclear translocation of CDK1/cyclin B1 complex, and increased MAD2 degradation, whereas CDK1 forced up-regulation partially rescues low cyclin B1 and MAD2 levels and G2-M transit in a TRAP1-poor background. Consistently, the CDK1 inhibitor RO-3306 is less active in a TRAP1-high background. Finally, a significant correlation was observed between TRAP1 and Ki67, CDK1 and/or MAD2 expression in breast, colorectal, and lung human tumour specimens. This study represents the first evidence that TRAP1 is relevant in the control of the complex machinery that governs cell cycle progression and mitotic entry and provides a strong rationale to regard TRAP1 as a biomarker to select tumours with deregulated cell cycle progression and thus likely poorly responsive to novel cell cycle inhibitors. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. The hypothesis that TRAP1 plays a regulatory function on the BRAF pathway, arising from the observation that BRAF levels are decreased upon TRAP1 interference, was tested in human breast and colorectal carcinoma in vitro and in vivo. This study shows that TRAP1 is involved in the regulation of BRAF synthesis/ubiquitination, without affecting its stability. Indeed, BRAF synthesis is facilitated in a TRAP1-rich background, whereas increased ubiquitination occurs upon disruption of the TRAP1 network that correlates with decreased protein levels. Remarkably, BRAF downstream pathway is modulated by TRAP1 regulatory activity: indeed, TRAP1 silencing induces (i) ERK phosphorylation attenuation, (ii) cell-cycle inhibition with cell accumulation in G0–G1 and G2–M transitions, and (iii) extensive reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-knockdown cells identified cell growth and cell-cycle regulation as the most significant biofunctions controlled by the TRAP1 network. It is worth noting that TRAP1 regulation on BRAF is conserved in human colorectal carcinomas, with the two proteins being frequently coexpressed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed activity against the TRAP1 network and high cytostatic potential in BRAF-mutated colorectal carcinoma cells. Therefore, this novel TRAP1 function represents an attractive therapeutic window to target dependency of BRAF-driven tumors on TRAP1 translational/quality control machinery. Cancer Res; 74(22); 6693–704. ©2014 AACR.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a heat shock protein 90 (HSP90) molecular chaperone upregulated in several human malignancies and involved in protection from apoptosis and drug resistance, cell cycle progression, cell metabolism and quality control of specific client proteins. TRAP1 role in thyroid carcinoma (TC), still unaddressed at present, was investigated by analyzing its expression in a cohort of 86 human TCs and evaluating its involvement in cancer cell survival and proliferationin vitro. Indeed, TRAP1 levels progressively increased from normal peritumoral thyroid gland, to papillary TCs (PTCs), follicular variants of PTCs (FV-PTCs) and poorly differentiated TCs (PDTCs). By contrast, anaplastic thyroid tumors exhibited a dual pattern, the majority being characterized by high TRAP1 levels, while a small subgroup completely negative. Consistently with a potential involvement of TRAP1 in thyroid carcinogenesis, TRAP1 silencing resulted in increased sensitivity to paclitaxel-induced apoptosis, inhibition of cell cycle progression and attenuation of ERK signaling. Noteworthy, the inhibition of TRAP1 ATPase activity by pharmacological agents resulted in attenuation of cell proliferation, inhibition of ERK signaling and reversion of drug resistance. These data suggest that TRAP1 inhibition may be regarded as potential strategy to target specific features of human TCs, i.e., cell proliferation and resistance to apoptosis.

Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro. Results: Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur. Conclusions: Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription. Mol Cancer Res; 10(2); 230–8. ©2011 AACR.

Epidermal growth factor receptor 1 (EGFR1) signaling is involved in human cancer cell progression and is responsible for aggressive biological behavior and poor clinical outcome in several human malignancies. Activation of the EGFR1 pathway has been proposed, among others, as being involved in the progression of thyroid cancer toward a thyroid-stimulating hormone (TSH)-independent phenotype. We have previously observed that undifferentiated thyroid carcinoma cells are hyper-sensitive to EGF signaling of downstream intracellular pathways, and this correlated both with the loss of TSH-dependency and increase in EGF-dependent proliferation and migration. Thus, we hypothesized that the upregulation of EGFR1 protein expression may be enhanced in parallel with transition toward a poorly differentiated phenotype in human thyroid carcinomas.The expression of EGFR1 was evaluated, by immunohistochemistry, in a series of 49 human thyroid carcinomas at different degrees of tumor differentiation.The expression of EGFR1 protein was significantly upregulated in poorly differentiated and anaplastic thyroid carcinomas, whereas it was absent or faint in normal thyroid gland tissue and in differentiated thyroid papillary carcinomas. Of note, selected thyroid tumors characterized by a mixed population of differentiated and undifferentiated tumor cells, likely progressing from well to poorly differentiated and anaplastic phenotypes, exhibited EGFR1-negative differentiated fields together with EGFR1-positive poorly differentiated and anaplastic areas.Upregulation of EGFR1 expression may be a molecular marker of dedifferentiation in thyroid epithelial carcinomas, likely being responsible for the activation of EGF signaling observed in tumor cells and favoring progression toward an angiogenic, poorly differentiated, TSH-independent phenotype.

Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called 'dysplastic' nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7+/-0.7) to vertical growth phase (3.6+/-3.1) to metastases (7.0+/-7.0) (P0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.

Objectives: Preoperative chemoradiation is currently the standard of care in locally advanced rectal carcinoma, even though a subset of rectal tumors does not achieve major clinically meaningful responses upon neoadjuvant chemoradiation. At present, no molecular biomarkers are available to predict response to neoadjuvant chemoradiation and select resistant tumors willing more intense therapeutic strategies. Thus, BRAF mutational status was investigated for its role in favoring resistance to radiation in colorectal carcinoma cell lines and cyclin-dependent kinase 1 as a target to improve radiosensitivity in BRAF V600E colorectal tumor cells. Methods: Colony-forming assay and apoptotic rates were evaluated to compare the sensitivity of different colon carcinoma cell lines to ionizing radiation and their radiosensitivity upon exposure to BRAF and/or cyclin-dependent kinase 1 inhibitory/silencing strategies. Cyclin-dependent kinase 1 expression/subcellular distribution was studied by immunoblot analysis. Results: Colon carcinoma BRAF V600E HT29 cells exhibited poor response to radiation compared to BRAF wild-type COLO320 and HCT116 cells. Interestingly, neither radiosensitizing doses of 5-fluoruracil nor BRAF inhibition/silencing significantly improved radiosensitivity in HT29 cells. Of note, poor response to radiation correlated with upregulation/relocation of cyclin-dependent kinase 1 in mitochondria. Consistently, cyclin-dependent kinase 1 inhibition/silencing as well as its targeting, through inhibition of HSP90 quality control pathway, significantly inhibited the clonogenic ability and increased apoptotic rates in HT29 cells upon exposure to radiation. Conclusion: These data suggest that BRAF V600E colorectal carcinoma cells are poorly responsive to radiation, and cyclin-dependent kinase 1 represents a target to improve radiosensitivity in BRAF V600E colorectal tumor cells.

BACKGROUND Prostate carcinomas are androgen-dependent neoplasms which progress toward a hormone-independent phenotype during hormone-deprivation therapy. We evaluated nevirapine, a reverse transcriptase inhibitor, as a new treatment in hormone-refractory prostate carcinoma cells with the aim of restoring the androgen-dependency of tumor cells, the rationale being that endogenous reverse transcriptase is up-regulated in transformed cells and reverse transcriptase inhibitors exert a differentiating activity in human tumors. METHODS AND RESULTS Nevirapine induced extensive reprogramming of gene expression in vitro with up-regulation of genes that might be silenced during prostate tumor progression (i.e., K18, PSA and androgen receptor) and down-regulation of genes involved in the progression toward an androgen-independent phenotype (i.e., K5, EGFR1, EGF and VEGF-A). Furthermore, nevirapine down-regulated the growth of prostate carcinoma xenografts in athymic mice and induced a differentiated phenotype in vivo with increased K18 expression. Interestingly, the drug restored androgen signaling by enhancing the ability of tumor cells to respond to dihydrotestosterone stimulation and to the antiproliferative activity of the androgen receptor blocker bicalutamide. Finally, nevirapine pretreatment increased the susceptibility of tumor cells to docetaxel, by enhancing their ability to undergo apoptosis. CONCLUSIONS These data suggest that nevirapine may be clinically tested in human hormone-refractory prostate carcinoma to restore the susceptibility to androgen deprivation therapy or to docetaxel. Prostate 69: 744–754, 2009. © 2009 Wiley-Liss, Inc.

Background: Nevirapine (NVP) is a nonnucleoside reverse transcriptase inhibitor used in HIV patients and recently evaluated as a differentiating and antiproliferative agent in human malignancies. However, while NVP is a safe treatment in immunocompromised patients, NVP-containing regimens have been associated with severe immune-mediated toxicities in non-HIV individuals. Methods and Results: We describe the toxicity profile of single-agent NVP in 6 non-HIV cancer patients treated for a median period of 7.3 months (range 1–24), reporting only a reversible grade III increase in glutamyl transpeptidase and glutamic pyruvic transaminase serum values. Interestingly, NVP treatment correlates with either a decrease in CD8+ T cell counts or a parallel increase in CD4/CD8 ratio, antithyroglobulin and antithyroid peroxidase autoantibody titers. Conclusions: These results, although obtained in a small cohort of patients, suggest that the toxicity profile of single-agent NVP may be worth testing in a phase I/II study in non-HIV cancer patients and that NVP toxicity may depend on its capacity to trigger autoimmune responses in susceptible individuals.

Two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, encode for endogenous nontelomeric reverse transcriptase (RT), a gene that is down-regulated in differentiated cells but is highly expressed in embryonic and transformed tissues. Two nonnucleosidic RT inhibitors, efavirenz and nevirapine, currently used in HIV treatment, reversibly down-regulate tumor growth and induce differentiation in several human tumor cell models.Aggressive biological behavior and loss of specific thyroid cell functions, such as thyroglobulin, thyroid peroxidase, TSH receptor, Na/I symporter expression, and iodine uptake are features of anaplastic thyroid cancer. Thus, we evaluated the use of RT inhibitors as a potentially differentiating and molecular-targeted treatment of this neoplasm.Our findings showed that nevirapine and efavirenz reversibly inhibit cell proliferation without triggering cell death in the undifferentiated thyroid carcinoma ARO and FRO cells, which exhibited high levels of endogenous RT activity. Inhibition of cell growth was correlated with accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the S phase. Moreover, treated cells demonstrated a differentiated phenotype and a significant reprogramming of gene expression characterized by up-regulation of the TSH receptor, thyroglobulin, thyroid peroxidase, and Na/I symporter genes. Interestingly, RT inhibition reestablished the ability to uptake iodine in response to TSH either in vitro or in vivo and reversibly down-regulated tumor growth in mice xenografts of ARO cells.These findings support the need for clinical trials to clarify whether RT inhibitors may restore the sensitivity to radiometabolic therapy in anaplastic thyroid tumors.

Survivin is involved in human cancer and is responsible for aggressive biological behavior and poor clinical outcomes in several human malignancies. Thus, we hypothesized that the upregulation of survivin protein expression may be enhanced in parallel with transition toward a poorly differentiated phenotype in human thyroid carcinomas.The expression of survivin was evaluated, using a standard linked streptavidin-biotin horseradish peroxidase technique technique, in a series of 56 human thyroid carcinomas (42 papillary, 4 poorly differentiated, and 10 anaplastic carcinomas) and thyroid carcinoma cell lines at different degrees of differentiation.The cytoplasmic expression of survivin protein was significantly upregulated in all thyroid tumors. A statistically significant association was found between nuclear survivin expression and anaplastic thyroid cancer (mean ± SD: well-differentiated thyroid cancer, 1.22 ± 20.21; non-well-differentiated thyroid cancer, 34.00 ± 25.17; anaplastic thyroid cancer, 56.50 ± 22.10; p0.001). Nuclear staining of survivin has been shown in poorly differentiated and anaplastic thyroid carcinomas, and this is likely due to the upregulation of the ΔEx3 survivin splicing variant, as shown in poorly differentiated/anaplastic thyroid carcinoma cell lines. Of note, selected thyroid tumors characterized by a mixed population of differentiated and undifferentiated neoplastic cells, likely progressing from well to poorly differentiated and anaplastic phenotypes, exhibited cytoplasmic expression of survivin in differentiated fields and nuclear protein staining in poorly differentiated and anaplastic areas. This expression profile provides substantial added value to conventional clinical markers in predicting anaplastic cancer. The cut-off for distinguishing thyroids that developed ATC from those that remained differentiated was30% of nuclear survivin expression. The receiver operating characteristic (ROC) area was 0.92, with a p-value of0.0001.Upregulation of survivin expression may be a molecular marker of dedifferentiation in thyroid epithelial carcinomas, likely being responsible for survival responses of tumor cells and, thus, favoring progression toward a poorly differentiated phenotype.

Sunitinib is currently being evaluated in advanced human thyroid carcinomas, based on the rationale that the vascular endothelial growth factor and platelet-derived growth factor receptors and the RET/PTC rearrangement are valuable targets for the treatment of this malignancy. However, criteria for selecting thyroid tumors that may benefit from sunitinib are lacking.The effect of activating somatic mutations in the KRAS and BRAF genes on the responsiveness to sunitinib was evaluated in a panel of thyroid cancer cell lines harboring wild-type KRAS and BRAF genes, the RET/PTC1 rearrangement, the G12R KRAS, or the V600E BRAF mutation.Sunitinib was found to selectively inhibit cell proliferation, induce cell accumulation in the G0-G1 phase, and inhibit the phosphorylation of ERK1/2 in both KRAS/BRAF wild-type thyroid cancer cells and in tumor cells harboring the RET/PTC rearrangement, whereas it was completely ineffective in KRAS- or BRAF-mutated thyroid carcinoma cells. This differential antitumor activity of sunitinib did not correlate with the expression profile of the vascular endothelial growth factor receptors 1, 2, and 3, platelet-derived growth factor receptor-α and cKIT genes. Of note, the constitutive activation of RAS/RAF/ERK signaling in KRAS/BRAF wild-type cells by transfection of the R12 HRAS or V600E BRAF mutants or stimulation with epithelial growth factor resulted in the loss of responsiveness to sunitinib, whereas pharmacological inhibition of MAPK kinase activity resulted in the resensitization of KRAS- or BRAF-mutated cells to the multikinase inhibitor.The constitutive activation of the RAS/RAF/ERK pathway may favor resistance to sunitinib in thyroid carcinoma cells.

The Ca2+-binding protein sorcin regulates intracellular calcium homeostasis and plays a role in the induction of drug resistance in human cancers. Recently, an 18 kDa mitochondrial isoform of sorcin was reported to participate in antiapoptosis in human colorectal cancer (CRC), but information remains lacking about the functional role of the more abundant 22 kDa isoform of sorcin expressed in CRC. We found the 22 kDa isoform to be widely expressed in human CRC cells, whether or not they were drug resistant. Its upregulation in drug-sensitive cells induced resistance to 5-fluorouracil, oxaliplatin, and irinotecan, whereas its downregulation sensitized CRC cells to these chemotherapeutic agents. Sorcin enhances the accumulation of Ca2+ in the endoplasmic reticulum (ER), preventing ER stress, and, in support of this function, we found that the 22 kDa isoform of sorcin was upregulated under conditions of ER stress. In contrast, RNAi-mediated silencing of sorcin activated caspase-3, caspase-12, and GRP78/BiP, triggering apoptosis through the mitochondrial pathway. Our findings establish that CRC cells overexpress sorcin as an adaptive mechanism to prevent ER stress and escape apoptosis triggered by chemotherapeutic agents, prompting its further investigation as a novel molecular target to overcome MDR. Cancer Res; 71(24); 7659–69. ©2011 AACR.

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group's use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1's presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER–mitochondria crosstalk.

TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca2+-binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway. Cancer Res; 70(16); 6577–86. ©2010 AACR.

The therapeutic role of EGFR inhibitors in thyroid malignancies is still controversial even though the full activation of EGF signaling has recently been proposed as involved in the dedifferentiation of human thyroid cancers.Agents which target EGFR signaling (erlotinib, cetuximab and panitumumab) were evaluated at preclinical level in a panel of thyroid tumor cell lines.Erlotinib induced a dose-dependent inhibition of cell proliferation together with inhibition of EGF-induced AKT and ERK1/2 signaling only in poorly-differentiated thyroid carcinoma FRO cells. By contrast, anti-EGFR monoclonal antibodies were inactive. Of note, erlotinib enhanced the proapoptotic activity of doxorubicin and paclitaxel and exhibited synergy with paclitaxel in poorly-differentiated thyroid carcinoma cells.EGFR signaling may represent a molecular target only in poorly-differentiated thyroid carcinoma cells, and agents that inhibit EGFR tyrosine kinase may be more effective than monoclonal antibodies which target the extracellular domain of the receptor.

TRAP1 is a component of a pro-survival mitochondrial pathway up-regulated in tumor cells. The evaluation of TRAP1 expression in 26 human colorectal carcinomas showed up-regulation in 17/26 tumors. Accordingly, TRAP1 levels were increased in HT-29 colorectal carcinoma cells resistant to 5-fluorouracil, oxaliplatin and irinotecan. Thus, we investigated the role of TRAP1 in multi-drug resistance in human colorectal cancer. Interestingly, TRAP1 overexpression leads to 5-fluorouracil-, oxaliplatin- and irinotecan-resistant phenotypes in different neoplastic cells. Conversely, the inhibition of TRAP1 activity by TRAP1 ATPase antagonist, shepherdin, increased the sensitivity to oxaliplatin and irinotecan in colorectal carcinoma cells resistant to the single agents. These results suggest that the increased expression of TRAP1 could be part of a pro-survival pathway responsible for multi-drug resistance. © 2009 Elsevier Ireland Ltd. All rights reserved.

Reverse transcriptase (RT) inhibitors are emerging as a novel class of anticancer differentiating agents, active in several human tumor cell models, such as melanoma and prostate, thyroid and colon carcinoma. Indeed, much evidence suggests that they may act by inhibiting endogenous RT, a gene highly expressed in undifferentiated and transformed cells. We therefore evaluated whether endogenous RT may represent a new molecular target in the treatment of human renal clear-cell carcinoma, a neoplasm with very low sensitivity to standard pharmacological therapies. Efavirenz and nevirapine, 2 non-nucleosidic RT inhibitors commonly used in HIV patients, either induced a reversible downregulation of cell proliferation or enhanced cell differentiation in primary cultures of human renal carcinoma cells characterized by high levels of endogenous RT activity. Both agents upregulated the expression of the vitamin D receptor and calbindin 28k genes, which are constitutively expressed in renal tubular cells, and induced vitamin D signaling by enhancing the ability of tumor cells to upregulate the vitamin D-dependent gene, CYP24. Furthermore, efavirenz- and nevirapine-differentiated tumor cells exhibited an immunogenic phenotype with an increased expression of HLA-I and CD40 antigens and an enhanced ability to elicit a specific T-cell response in mixed lymphocyte/tumor-cell cultures. Indeed, renal carcinoma cells exposed to efavirenz induced a CD8+CCR7-CD45RA− effector memory T-cell phenotype, whereas untreated RCC cells induced a CD8+CCR7+CD45RA− central memory T-cell phenotype. These data suggest that RT inhibitors may be a novel tool in the treatment of human renal clear-cell carcinoma, potentially able to enhance the immunogenic potential of tumor cell. © 2008 Wiley-Liss, Inc.

Colorectal cancer is one of the leading causes of cancer deaths worldwide. For decades, 5-fluorouracil has been the only effective chemotherapeutic agent for the treatment of this disease. Lately, the addition of irinotecan and oxaliplatin to standard 5-fluorouracil-based chemotherapy has improved not only response rates but also overall survival. Intense efforts have also focused on identifying novel agents targeting specific growth factor receptors, such as epidermal growth factor receptor 1 (EGFR1), which is over-expressed in a number of solid tumours of ectodermal origin, including colon adenocarcinoma, or mediators of angiogenesis, such as vascular endothelial growth factor (VEGF). Some of these molecular-targeted agents (i.e. the anti-EGFR1 monoclonal antibodies cetuximab and panitumumab, and the anti-VEGF monoclonal antibody bevacizumab) can be safely and effectively used in combination with conventional chemotherapy in colorectal cancer patients. However, recent evidence suggests that anti-EGFR1 antibodies are ineffective in tumours bearing the oncogenic activation of signalling pathways downstream of the EGFR1. Therefore, in spite of these advances, there is still a strong need for more effective and well-tolerated therapies for the management of human colorectal carcinoma. With such a perspective, this review addresses some of the critical issues under evaluation in either pre-clinical studies or clinical trials and focuses on innovative strategies for the rational combination of anticancer drugs in order to improve the synergism between traditional chemotherapeutics and novel molecular-targeted agents. Based on the evidence that colorectal cancer is characterised by the activation of survival signals responsible for protecting cells from the cytotoxic effects of anticancer agents, we discuss the role of the TNF-receptor-associated protein 1 (TRAP1)/heat shock protein 90 (HSP90) anti-apoptotic pathway, which is involved in drug resistance and may became a novel potential molecular target to improve the efficacy of drug therapies in human colorectal carcinoma.