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Tay K.H.,University of Newcastle | Luan Q.,University of Newcastle | Croft A.,University of Newcastle | Croft A.,Oncology and Immunology Unit | And 4 more authors.
Cellular Signalling | Year: 2014

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells. © 2013 Elsevier Inc.

Fonsatti E.,University of Siena | Maio M.,University of Siena | Maio M.,Cancer Bioimmunotherapy Unit | Altomonte M.,University of Siena | Hersey P.,Oncology and Immunology Unit
Seminars in Oncology | Year: 2010

CD40 is a costimulatory molecule widely expressed by immune cells and by neoplastic cells of different histotypes. Engagement of surface CD40 mediates different effects depending on cell type and microenvironment. In particular, CD40 expression on immune cells regulates humoral and cellular immunity, while it has apoptotic and antiproliferative activity on selected neoplastic cells. Thus, CD40 targeting may indirectly affect tumor growth through the activation of immune cells and/or directly by mediating cytotoxic effects on neoplastic cells. Preliminary findings emerging from clinical trials indicate that antibodies to CD40 can induce immune modulation and clinical responses in cancer patients. © 2010 Elsevier Inc. All rights reserved.

Croft A.,University of Newcastle | Croft A.,Oncology and Immunology Unit | Tay K.H.,University of Newcastle | Boyd S.C.,University of Sydney | And 8 more authors.
Journal of Investigative Dermatology | Year: 2014

Cancer cells commonly undergo chronic endoplasmic reticulum (ER) stress, to which the cells have to adapt for survival and proliferation. We report here that in melanoma cells intrinsic activation of the ER stress response/unfolded protein response (UPR) is, at least in part, caused by increased outputs of protein synthesis driven by oncogenic activation of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) and promotes proliferation and protects against apoptosis induced by acute ER stress. Inhibition of oncogenic BRAF V600E or MEK-attenuated activation of inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) signaling of the UPR in melanoma cells. This was associated with decreased phosphorylation of eukaryotic initiation factor 4E (eIF4E) and nascent protein synthesis and was recapitulated by knockdown of eIF4E. In line with this, introduction of BRAF V600E into melanocytes led to increases in eIF4E phosphorylation and protein production and triggered activation of the UPR. Similar to knockdown of glucose-regulated protein 78 (GRP78), inhibition of XBP1 decelerated melanoma cell proliferation and enhanced apoptosis induced by the pharmacological ER stress inducers tunicamycin and thapasigargin. Collectively, these results reveal that potentiation of adaptation to chronic ER stress is another mechanism by which oncogenic activation of the MEK/ERK pathway promotes the pathogenesis of melanoma. © 2014 The Society for Investigative Dermatology.

Lai F.,University of Newcastle | Guo S.T.,Shanxi Cancer Hospital and Institute | Jin L.,University of Newcastle | Jiang C.C.,University of Newcastle | And 11 more authors.
Cell Death and Disease | Year: 2013

Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAFV600E melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAFV600E melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRA FV600E melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma. © 2013 Macmillan Publishers Limited All rights reserved.

Zhuang L.,Tissue Pathology and Diagnostic Oncology | Zhuang L.,Melanoma Institute Australia | Scolyer R.A.,Tissue Pathology and Diagnostic Oncology | Scolyer R.A.,Melanoma Institute Australia | And 11 more authors.
Modern Pathology | Year: 2010

The serum level of lactate dehydrogenase (LDH) is an important predictor of prognosis and treatment response in melanoma patients. It is unknown whether the expression of LDH-5 in tissue sections also has prognostic significance and whether it is related to the expression of the anti-apoptotic proteins, Bcl-2, Bcl-XL and Mcl-1, and endoplasmic reticulum stress protein glucose-regulated protein 78 (GRP78). Identification of an association between LDH-5 expression and anti-apoptotic proteins may have important therapeutic implications for melanoma patients. Sections from 159 pigmented lesions, including nevi and melanoma at different stages of progression were studied by immunohistochemistry. Correlation of LDH-5 expression with clinicopathological factors and with the expression of Bcl-2, Bcl-XL, Mcl-1 and GRP78 was examined. LDH-5 was detected at low levels in 6 of 10 compound nevi (60%) and 6 of 10 dysplastic nevi (60%). The percentage of positive cases was greater in thin (1.0 mm) (74%) and thick primary melanoma (1.0 mm) (95%) and in metastatic melanoma in the skin (100%) and lymph node (81%). The immunoreactive score was highly related to progression of melanoma (P0.0001). LDH-5 expression was positively associated with increasing tumor thickness (P0.02) and dermal tumor mitotic rate (P0.02). LDH-5 above the median immunoreactive score was associated with reduced disease-free survival and overall survival (P0.02). LDH-5 expression was negatively associated with Bcl-2 expression. In contrast, LDH-5 expression was strongly associated with Bcl-XL and Mcl-1 expression and also positively associated with GRP78 expression (P0.0001). The low Bcl-2 expression in melanomas with high LDH-5 expression provides an explanation for the poor response of patients with high serum LDH levels to treatment with the Bcl-2 antisense drug Genasense. The strong correlation of LDH-5 expression with Mcl-1 expression suggests that treatment strategies inhibiting the activity of Mcl-1 in melanoma patients should be investigated. © 2010 USCAP, Inc. All rights reserved.

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