Melinda M. Mortenson

Targeting BCL-2 overexpression in various human malignancies through NF-κB inhibition by the proteasome inhibitor bortezomib

BackgroundBCL-2 overexpression occurs in many cancer types and is associated with chemoresistance and radioresistance. The mechanisms responsible for its aberrant expression are thought to be transcriptionally mediated but remain unclear. We examined the cell type-specific mechanism of BCL-2 gene transcription in various solid organ malignancies.MethodsRegulation of BCL-2 gene transcription was examined in seven different human cancer cell lines including two pancreatic (MIA-PaCa-2, PANC-1), two prostate (LNCaP, PC-3), two lung (Calu-1, A549) and one breast (MCF-7) cancer cell line. Cells were treated with inhibitors of phosphatidylinositol-3 kinase (PI3K), MEK/ERK, and p38MAPK. The effect of mutation of a NF-κB site in the BCL-2 promoter was determined, as was the effect of inhibition of NF-κB function using a 26S proteasome inhibitor (bortezomib) on both BCL-2 transcription and induction of apoptosis.ResultsBCL-2 expression varied both between and within tumor types; four of seven cell lines demonstrated high BCL-2 levels (MIA-PaCa-2, PC-3, Calu-1 and MCF-7). No signaling pathway was uniformly responsible for overexpression of BCL-2; however, mutation of the NF-κB site decreased BCL-2 promoter activity in all cell lines. Inhibition of NF-κB activity decreased BCL-2 protein levels independently of the signaling pathway involved in transcriptional activation of the BCL-2 gene.ConclusionsDiverse signaling pathways variably regulate BCL-2 gene expression in a cell type-specific fashion. Therapy to decrease BCL-2 levels in various human cancers would be more broadly applicable if targeted to transcriptional activation rather than signal transduction cascades. Finally, the apoptotic efficacy of proteasome inhibition with bortezomib paralleled the ability to inhibit NF-κB activity and decrease BCL-2 levels.

Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapy in the A549 non-small-cell lung cancer cell line.

BackgroundNon-small-cell lung cancer (NSCLC) has a poor prognosis. Despite advances in therapy, survival has improved only slightly. The 26S proteasome regulates multiple cellular processes through degradation of ubiquitin-tagged proteins. The proteasome inhibitor, bortezomib (Velcade, formerly PS-341), has been shown to be an active anticancer agent both in vitro and in vivo in multiple tumor types.PurposeTo determine the molecular and cellular effects of the proteasome inhibitor in NSCLC as well as to evaluate the effectiveness of sequential treatment with bortezomib and gemcitabine/carboplatin (G/C) chemotherapy both in vitro and in vivo.MethodsAll experiments were performed in the A549 NSCLC cell line. MTT assays were used to evaluate cytotoxicity. Western blotting evaluated protein levels. Measures of apoptosis included FACS analysis, DAPI staining and caspase-3 cleavage. Long-term cell viability was determined using an anchorage-dependent clonogenic assay. Sequential studies were performed in vitro and in vivo.ResultsBortezomib increased p21waf1/cip1, induced G2/M arrest, and triggered a small amount of apoptosis. The apoptotic effect of G/C chemotherapy was eliminated when bortezomib was administered prior to the chemotherapy; however, it was accentuated when the bortezomib was given simultaneously or after the chemotherapy.ConclusionsBortezomib improves efficacy in combination with gemcitabine and carboplatin in NSCLC, but sequential effects are important and must be considered when developing therapeutic regimens.

ERK/BCL-2 pathway in the resistance of pancreatic cancer to anoikis.

BACKGROUND Anoikis is a special type of programmed cell death after loss of cell-cell and cell-extracellular matrix interactions. Resistance to anoikis is likely involved in the process of metastasis, specifically during the tumor cell migration through lymph or vascular channels. We have previously shown that BCL-2 confers resistance to other forms of programmed cell death (i.e., apoptosis); furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates BCL-2 expression. We therefore tested the hypothesis that pancreatic cancer cell lines are resistant to anoikis and this resistance is due to activation of ERK1/2 and subsequent overexpression of BCL-2. MATERIALS AND METHODS Pancreatic cancer cell lines (MIA-PaCa-2 and BxPC-3) were examined for cell death following loss of adherence to extracellular matrix. Subclones of the MIA-PaCa-2 cell line (either selected in vivo for increased metastatic potential [MIA-LM2] or overexpressing BCL-2 [MIA-BCL2]) were also examined for induction of anoikis following loss of extracellular matrix adherence. Finally, the effect of the ERK inhibitor (PD98059) on BCL-2 expression and induction of anoikis was examined. RESULTS Under conditions of loss of cell-extracellular matrix interaction, pancreatic cancer cells undergo varying amounts of anoikis. Basal levels of activated ERK and BCL-2 paralleled the sensitivity to induction of anoikis. The highly metastatic cell line, MIA-LM2, was more resistant to anoikis than the parental cell line. Inhibition of ERK down-regulated BCL-2 and was associated with restoration of sensitivity to anoikis. CONCLUSIONS Activation of a signaling pathway from ERK to overexpression of BCL-2 may confer resistance to anoikis, a critical step in the development of metastasis. Targeting the ERK/BCL-2 pathway may lead to sensitization of pancreatic cancer to anoikis, thereby decreasing the ability of these cells to metastasize.

BCL-2 functions as an activator of the AKT signaling pathway in pancreatic cancer

BCL‐2 is the prototypic anti‐apoptotic protein involved in the regulation of apoptosis. Overexpression of BCL‐2 is common in pancreatic cancer and confers resistance to the apoptotic effect of chemo‐ and radiotherapy. Although these cellular effects of BCL‐2 are traditionally related to pathways involving the mitochondrial membrane, we sought to investigate whether BCL‐2 is involved in other signaling pathways regulating cell survival and focused on AKT. We examined the effect of overexpression of BCL‐2 in the MIA‐PaCa‐2 human pancreatic cancer cell line on the function and subcellular location of AKT. We observed that the stable subclones of MIA‐PaCa‐2 overexpressing BCL‐2 demonstrated increased activity of AKT as well as IKK (a downstream target of AKT), increasing the transcriptional activity of NF‐κB. Using immunoprecipitation techniques, we observed co‐immunoprecipitation of AKT and BCL‐2. Immunocytochemistry demonstrated co‐localization of BCL‐2 and AKT, which was abrogated by treatment with HA14‐1, a small molecule inhibitor of BH‐3‐mediated protein interaction by BCL‐2. Furthermore, treatment with HA14‐1 decreased phosphorylation of AKT and increased sensitivity to the apoptotic effect of the chemotherapeutic agent, paclitaxel. These results demonstrate an additional mechanism of regulation of cell survival mediated by BCL‐2, namely through AKT activation, in the MIA‐PaCa‐2 pancreatic cancer cell line. Therefore, directed inhibition of BCL‐2 may alter diverse pathways controlling cell survival and overcome the apoptotic resistance that is the hallmark of pancreatic cancer. J. Cell. Biochem. 102: 1171–1179, 2007.

Symptomatic pancreatic polypeptide-secreting tumor of the distal pancreas (PPoma)

Our report describes a 46-yr-old woman who presented with watery diarrhea in the presence of multiple endocrine neoplasia type I (MEN I) syndrome. Of various potential pancreatic endocrine hormones, only serum levels of pancreatic polypeptide were elevated. Radiologic imaging failed to identify a pancreatic tumor; her diarrhea was therefore managed with subcutaneous administration of somatostatin. Three years later she developed gallstone pancreatitis with the subsequent development of a pancreatic pseudocyst. At exploration for drainage of the pseudocyst, intraoperative ultrasound identified a 6-mm tumor in the distal pancreas that was resected. Final pathology documented a pancreatic endocrine tumor with immunohistochemical staining demonstrating the presence of pancreatic polypeptide. The present case illustrates the symptomatology that may be associated with pancreatic polypeptide-secreting endocrine tumors of the pancreas.