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Marzese D.M.,John Wayne Cancer Institute | Scolyer R.A.,Royal Prince Alfred Hospital | Scolyer R.A.,University of Sydney | Scolyer R.A.,Melanoma Institute Australia | And 14 more authors.
Neuro-Oncology | Year: 2014

Background: The brain is a common target of metastases for melanoma patients. Little is known about the genetic and epigenetic alterations in melanoma brain metastases (MBMs). Unraveling these molecular alterations is a key step in understanding their aggressive nature and identifying novel therapeutic targets. Methods: Genome-wide DNA methylation analyses of MBMs (n = 15) and normal brain tissues (n = 91) and simultaneous multigene DNA methylation and gene deletion analyses of metastatic melanoma tissues (99 MBMs and 43 extracranial metastases) were performed. BRAF and NRAS mutations were evaluated in MBMs by targeted sequencing. Results: MBMs showed significant epigenetic heterogeneity. RARB, RASSF1, ESR1, APC, PTEN, and CDH13 genes were frequently hypermethylated. Deletions were frequently detected in the CDKN2A/B locus. Of MBMs, 46.1% and 28.8% had BRAF and NRAS missense mutations, respectively. Compared with lung and liver metastases, MBMs exhibited higher frequency of CDH13 hypermethylation and CDKN2A/B locus deletion. Mutual exclusivity between hypermethylated genes and CDKN2A/B locus deletion identified 2 clinically relevant molecular subtypes of MBMs. CDKN2A/B deletions were associated with multiple MBMs and frequently hypermethylated genes with shorter time to brain metastasis. Conclusions: Melanoma cells that colonize the brain harbor numerous genetically and epigenetically altered genes. This study presents an integrated genomic and epigenomic analysis that reveals MBM-specific molecular alterations and mutually exclusive molecular subtypes. © The Author(s) 2014.


Flamini M.I.,Institute of Medicine and Experimental Biology of Cuyo | Gauna G.V.,Institute of Medicine and Experimental Biology of Cuyo | Gauna G.V.,National University of Cuyo | Sottile M.L.,Institute of Medicine and Experimental Biology of Cuyo | And 4 more authors.
Journal of Cellular and Molecular Medicine | Year: 2014

Breast cancer is the most common malignancy in women and the appearance of distant metastases produces the death in 98% of cases. The retinoic acid receptor β (RARβ) is not expressed in 50% of invasive breast carcinoma compared with normal tissue and it has been associated with lymph node metastasis. Our hypothesis is that RARβ protein participates in the metastatic process. T47D and MCF7 breast cancer cell lines were used to perform viability assay, immunobloting, migration assays, RNA interference and immunofluorescence. Administration of retinoic acid (RA) in breast cancer cells induced RARβ gene expression that was greatest after 72 hrs with a concentration 1 μM. High concentrations of RA increased the expression of RARβ causing an inhibition of the 60% in cell migration and significantly decreased the expression of migration-related proteins [moesin, c-Src and focal adhesion kinase (FAK)]. The treatment with RARα and RARγ agonists did not affect the cell migration. On the contrary, the addition of the selective retinoid RARβ-agonist (BMS453) significantly reduced cell migration comparable to RA inhibition. When RARβ gene silencing was performed, the RA failed to significantly inhibit migration and resulted ineffective to reduce moesin, c-Src and FAK expressions. RARβ is necessary to inhibit migration induced by RA in breast cancer cells modulating the expression of proteins involved in cell migration. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.


Nadin S.B.,Institute of Medicine and Experimental Biology of Cuyo | Sottile M.L.,Institute of Medicine and Experimental Biology of Cuyo | Montt-Guevara M.M.,Institute of Medicine and Experimental Biology of Cuyo | Gauna G.V.,Institute of Medicine and Experimental Biology of Cuyo | And 8 more authors.
Cell Stress and Chaperones | Year: 2014

Neoadjuvant chemotherapy is used in patients with locally advanced breast cancer to reduce tumor size before surgery. Unfortunately, resistance to chemotherapy may arise from a variety of mechanisms. Heat shock proteins (HSPs), which are highly expressed in mammary tumor cells, have been implicated in anticancer drug resistance. In spite of the widely described value of HSPs as molecular markers in cancer, their implications in breast tumors treated with anthracycline-based neoadjuvant chemotherapy has been poorly explored. In this study, we have evaluated, by immunohistochemistry, the expression of HSP27 (HSPB1) and HSP70 (HSPA) in serial biopsies from locally advanced breast cancer patients (n=60) treated with doxorubicin (DOX)- or epirubicin (EPI)-based monochemotherapy. Serial biopsies were taken at days 1, 3, 7, and 21, and compared with prechemotherapy and surgical biopsies. After surgery, the patients received additional chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil. High nuclear HSPB1 and HSPA expressions were found in invasive cells after DOX/EPI administration (P<0.001), but the drug did not affect the cytoplasmic expression of the HSPs. Infiltrating lymphocytes showed high nuclear HSPA (P<0.01) levels at postchemotherapy. No correlations were found between HSPs expression and the clinical and pathological response to neoadjuvant therapy. However, in postchemotherapy biopsies, high nuclear (>31 % of the cells) and cytoplasmic HSPA expressions (>11 % of the tumor cells) were associated with better DFS (P=0.0348 and P=0.0118, respectively). We conclude that HSPA expression may be a useful prognostic marker in breast cancer patients treated with neoadjuvant DOX/EPI chemotherapy indicating the need to change the administered drugs after surgery for overcoming drug resistance. © 2013 Cell Stress Society International.


Sottile M.L.,Institute of Medicine and Experimental Biology of Cuyo | Losinno A.D.,Institute of Histology and Embryology | Losinno A.D.,National University of Cuyo | Fanelli M.A.,Institute of Medicine and Experimental Biology of Cuyo | And 5 more authors.
International Journal of Hyperthermia | Year: 2015

Purpose: Hyperthermia is used in combination with conventional anticancer agents to potentiate their cytotoxicity. One of its key events is the synthesis of heat shock proteins (HSPs), which are able to associate with components from DNA repair mechanisms. However, little is known about their relationship with the mismatch repair system (MMR). Our aim was to study the effects of hyperthermia on cisplatin (cPt) sensitivity and to determine whether MLH1 and MSH2 associate with Hsp27 and Hsp72 in MMR-deficient(-)/-proficient(+) cells.Materials and methods: HCT116+ch2 (MMR-) and HCT116+ch3 (MMR+) cell lines were exposed to cPt with or without previous hyperthermia (42 °C, 1 h). Clonogenic survival assays, MTT, confocal immunofluorescence, immunoprecipitation, immunoblotting and flow cytometry were performed.Results: Hyperthermia increased the cPt resistance in MMR- cells 1.42-fold. Immunofluorescence revealed that after cPt, Hsp27 and Hsp72 translocated to the nucleus and colocalisation coefficients between these proteins with MLH1 and MSH2 increased in MMR+ cells. Immunoprecipitation confirmed the interactions between HSPs and MMR proteins in control and treated cells. Hyperthermia pretreatment induced cell cycle arrest, increased p73 expression and potentiated cPt sensitivity in MMR+ cells.Conclusions: This is the first report showing in a MMR-/+ cellular model that MLH1 and MSH2 are client proteins of Hsp27 and Hsp72. Our study suggests that p73 might participate in the cellular response to hyperthermia and cPt in a MMR-dependent manner. Further functional studies will confirm whether HSPs cooperate with the MMR system in cPt-induced DNA damage response or whether these protein interactions are only the result of their chaperone functions. © 2015 Informa UK Ltd. All rights reserved.


Nadin S.B.,Institute of Medicine and Experimental Biology of Cuyo | Cuello-Carrion F.D.,Institute of Medicine and Experimental Biology of Cuyo | Sottile M.L.,Institute of Medicine and Experimental Biology of Cuyo | Ciocca D.R.,Institute of Medicine and Experimental Biology of Cuyo | And 2 more authors.
International Journal of Hyperthermia | Year: 2012

Purpose: The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs). Materials and methods: The human colon carcinoma cell lines HCT116 (parent cells), HCT116+ch2 (MMR-deficient), and HCT116+ch3 (MMR-proficient) were used. Cells were incubated at 41°C and 42°C for 1h and then at 37°C for 4 and 24h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage. Results: The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116+ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116+ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate. Conclusions: The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116+ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells. © 2012 Informa UK Ltd All rights reserved.


PubMed | Institute of Medicine and Experimental Biology of Cuyo
Type: Journal Article | Journal: Cell stress & chaperones | Year: 2014

Neoadjuvant chemotherapy is used in patients with locally advanced breast cancer to reduce tumor size before surgery. Unfortunately, resistance to chemotherapy may arise from a variety of mechanisms. Heat shock proteins (HSPs), which are highly expressed in mammary tumor cells, have been implicated in anticancer drug resistance. In spite of the widely described value of HSPs as molecular markers in cancer, their implications in breast tumors treated with anthracycline-based neoadjuvant chemotherapy has been poorly explored. In this study, we have evaluated, by immunohistochemistry, the expression of HSP27 (HSPB1) and HSP70 (HSPA) in serial biopsies from locally advanced breast cancer patients (n=60) treated with doxorubicin (DOX)- or epirubicin (EPI)-based monochemotherapy. Serial biopsies were taken at days 1, 3, 7, and 21, and compared with prechemotherapy and surgical biopsies. After surgery, the patients received additional chemotherapy with cyclophosphamide, methotrexate, and 5-fluorouracil. High nuclear HSPB1 and HSPA expressions were found in invasive cells after DOX/EPI administration (P<0.001), but the drug did not affect the cytoplasmic expression of the HSPs. Infiltrating lymphocytes showed high nuclear HSPA (P<0.01) levels at postchemotherapy. No correlations were found between HSPs expression and the clinical and pathological response to neoadjuvant therapy. However, in postchemotherapy biopsies, high nuclear (>31% of the cells) and cytoplasmic HSPA expressions (>11% of the tumor cells) were associated with better DFS (P=0.0348 and P=0.0118, respectively). We conclude that HSPA expression may be a useful prognostic marker in breast cancer patients treated with neoadjuvant DOX/EPI chemotherapy indicating the need to change the administered drugs after surgery for overcoming drug resistance.


PubMed | Institute of Medicine and Experimental Biology of Cuyo
Type: Journal Article | Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group | Year: 2015

Hyperthermia is used in combination with conventional anticancer agents to potentiate their cytotoxicity. One of its key events is the synthesis of heat shock proteins (HSPs), which are able to associate with components from DNA repair mechanisms. However, little is known about their relationship with the mismatch repair system (MMR). Our aim was to study the effects of hyperthermia on cisplatin (cPt) sensitivity and to determine whether MLH1 and MSH2 associate with Hsp27 and Hsp72 in MMR-deficient(-)/-proficient(+) cells.HCT116+ch2 (MMR-) and HCT116+ch3 (MMR+) cell lines were exposed to cPt with or without previous hyperthermia (42C, 1h). Clonogenic survival assays, MTT, confocal immunofluorescence, immunoprecipitation, immunoblotting and flow cytometry were performed.Hyperthermia increased the cPt resistance in MMR- cells 1.42-fold. Immunofluorescence revealed that after cPt, Hsp27 and Hsp72 translocated to the nucleus and colocalisation coefficients between these proteins with MLH1 and MSH2 increased in MMR+ cells. Immunoprecipitation confirmed the interactions between HSPs and MMR proteins in control and treated cells. Hyperthermia pretreatment induced cell cycle arrest, increased p73 expression and potentiated cPt sensitivity in MMR+ cells.This is the first report showing in a MMR-/+ cellular model that MLH1 and MSH2 are client proteins of Hsp27 and Hsp72. Our study suggests that p73 might participate in the cellular response to hyperthermia and cPt in a MMR-dependent manner. Further functional studies will confirm whether HSPs cooperate with the MMR system in cPt-induced DNA damage response or whether these protein interactions are only the result of their chaperone functions.


PubMed | Institute of Medicine and Experimental Biology of Cuyo
Type: Journal Article | Journal: International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia Group | Year: 2012

The objective of the present study was to examine the consequences of a mild hyperthermia in human tumour cell lines deficient and proficient in the DNA mismatch repair system (MMR) to advance our understanding on the relationship between MMR and heat shock proteins (HSPs).The human colon carcinoma cell lines HCT116 (parent cells), HCT116+ch2 (MMR-deficient), and HCT116+ch3 (MMR-proficient) were used. Cells were incubated at 41C and 42C for 1h and then at 37C for 4 and 24h. The expression of Hsp27 and Hsp72 was evaluated by immunocytochemistry. Hsp27, Hsp72, hMLH1 and hMSH2 levels were assessed by western blotting in nuclear and cytoplasmic fractions. The alkaline comet assay was used to evaluate the DNA damage.The mild hyperthermia significantly increased the protein expression levels of Hsp27 and Hsp72 in all cell lines, which was higher in the cytoplasm and nucleus of HCT116+ch3 cells. We also observed that heat induced translocation of hMLH1 and hMSH2 proteins from the nucleus to the cytoplasm in HCT116+ch3 cells. The comet assay revealed that HCT116 parent cells were more resistant to heat-induced DNA damage. However, the MMR-proficient and deficient cell lines repaired the DNA damage at the same rate.The present study demonstrates that hyperthermia induced the nuclear accumulation of Hsp27 and Hsp72 and affected the subcellular localisation of hMLH1 and hMSH2 in HCT116+ch3 cells. Our findings suggest that the MMR system is not a direct determining factor for the different heat shock response in HCT116 cells.

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