Trastuzumab signaling in ErbB2-overexpressing inflammatory breast cancer correlates with X-linked inhibitor of apoptosis protein expression

Abstract

Inflammatory breast cancer (IBC) patients show poor survival and a significant incidence of epidermal growth factor receptor-2 (ErbB2) overexpression. A distinct mechanism involving increased expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin, key members of the inhibitor of apoptosis protein (IAP) family, was observed post-trastuzumab (an ErbB2 monoclonal antibody) treatment in an ErbB2-overexpressing, estrogen receptor negative, IBC cellular model, SUM190PT, isolated from a primary IBC tumor. In contrast, a decrease in the IAP expression was observed in the non-IBC, ErbB2-overexpressing SKBR3 cells in which trastuzumab treatment also decreased p-AKT and cell viability. Further, in SUM190PT cells, therapeutic sensitivity to GW583340 (a dual epidermal growth factor receptor/ErbB2 kinase inhibitor) corresponded with XIAP down-regulation and abrogation of XIAP inhibition on active caspase-9 release. Specific small interfering RNA -mediated XIAP inhibition in combination with trastuzumab caused decrease in inactive procaspase-9 and inhibition of p-AKT corresponding with 45% to 50% decrease in cell viability in the SUM190PT cells, which have high steady-state p-AKT levels. Further, embelin, a small-molecule inhibitor that abrogates binding of XIAP to procaspase-9, caused significant decrease in SUM190PT viability. However, embelin in combination with trastuzumab failed to affect SUM190PT viability because it has no direct effect on XIAP, which is induced by trastuzumab treatment. These data have identified a novel functional link between ErbB2 signaling and antiapoptotic pathway mediated by XIAP. Blockade of the IAP antiapoptotic pathway alone or in combination would be an attractive strategy in IBC therapy.

Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration

“…Anti-apoptotic signaling in IBC cells has been studied in the past, most prominently in the context of chemotherapeutic resistance. 39,40 A more recent example is the receptor tyrosine kinase Axl, which has recently been shown to have a significant role in the malignant behavior of IBC cells 16 and has been previously revealed to block the induction of apoptosis through Akt and Bcl-xL. 41 However, our study is the first (to our knowledge) to directly address the molecular mechanisms that allow IBC cell survival in the context of ECM detachment.…”

MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition

“…Cells at 50% confluence in a 96-well plate (Corning Incorporated, Corning, NY, USA) were treated with 0 -200 mmol l À1 cisplatin (Sigma Chemical Co., St Louis, MO, USA) for 24 h in a serum-containing medium. Cell were then incubated at 371C for 2 h in a medium containing 0.5 g l À1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co.), and a proliferation assay was conducted as reported earlier (Amantana et al, 2004;Aird et al, 2008). In some experiments, cell viability was also determined by Trypan blue exclusion assay (Aird et al, 2008).…”

“…Cells were harvested and immediately lysed in an NP40 cell lysis buffer (Biosource, Carlsbad, CA, USA), subjected to SDS -PAGE under reducing conditions and transferred as reported earlier (Aird et al, 2008). The membranes were probed overnight with a primary antibody against XIAP (1 : 1000 dilution; BD Transduction Laboratories, Lexington, KY, USA), PKC-d (1 : 1000; Cell Signaling Technology, Beverly, MA, USA), procaspase-9 (1 : 1000, Neomarker, Fremont, CA, USA), MLH1, PMS2 and MSH6 (1 : 500, BD Pharmingen, Lexington, KY, USA) at 41C.…”

“…The membranes were probed overnight with a primary antibody against XIAP (1 : 1000 dilution; BD Transduction Laboratories, Lexington, KY, USA), PKC-d (1 : 1000; Cell Signaling Technology, Beverly, MA, USA), procaspase-9 (1 : 1000, Neomarker, Fremont, CA, USA), MLH1, PMS2 and MSH6 (1 : 500, BD Pharmingen, Lexington, KY, USA) at 41C. Immunoreactive bands were visualised as reported earlier (Aird et al, 2008). b-Actin or GAPDH served as loading control by re-probing the same membrane with an antibody (1 : 4000; Calbiochem, La Jolla, CA, USA) or probed after first stripping the membrane in a stripping buffer (100 mmol l À1 2-mercaptoethanol-2% SDS-62.5 mmol l À1 Tris-HCl (pH 6.7)) at 501C for 30 min, followed by a washing and blocking procedure as described above.…”

“…The X-linked inhibitor of apoptosis protein (XIAP) is considered to be one of the most potent IAPs and a versatile caspase inhibitor (Deveraux et al, 1997;Devi, 2004). Multiple studies in EOC (Sasaki et al, 2000;Asselin et al, 2001;Li et al, 2001;Mansouri et al, 2003;Yang et al, 2005) and other cancers (Amantana et al, 2004;Schimmer et al, 2006;Aird et al, 2008) have identified XIAP suppression to be a key mechanism in chemotherapy resistance. The acquisition of cisplatin resistance is associated with the ability of treated cells to upregulate XIAP expression.…”