Michael G. Schlieman

Incidence, mechanism and prognostic value of activated AKT in pancreas cancer

When activated, the serine/threonine kinase AKT mediates an antiapoptotic signal implicated in chemoresistance of various cancers. The mechanism(s) of AKT activation are unknown, though overexpression of HER-2/neu has been implicated in breast cancer. Therefore, we determined the incidence of activated AKT in human pancreatic cancer, whether HER-2/neu is involved in AKT activation, and if AKT activation is associated with biologic behaviour. HER-2/neu expression and AKT activation were examined in seven pancreatic cancer cell lines by Western blotting. The in vitro effect of HER-2/neu inhibition on AKT activation was similarly determined. Finally, 78 pancreatic cancer specimens were examined for AKT activation and HER-2/neu overexpression, and correlated with the clinical prognostic variable of histologic grade. HER-2/neu was overexpressed in two of seven cell lines; these two cell lines demonstrated the highest level of AKT activation. Inhibition of HER-2/neu reduced AKT activation in vitro. AKT was activated in 46 out of 78 (59%) of the pancreatic cancers; HER-2/neu overexpression correlated with AKT activation (P=0.015). Furthermore, AKT activation was correlated with higher histologic tumour grade (P=0.047). Thus, it is concluded that AKT is frequently activated in pancreatic cancer; this antiapoptotic signal may be mediated by HER-2/neu overexpression. AKT activation is associated with tumour grade, an important prognostic factor.

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.

AKT inhibition is associated with chemosensitisation in the pancreatic cancer cell line MIA-PaCa-2

Activation of the serine/threonine kinase AKT is common in pancreatic cancer; inhibition of which sensitises cells to the apoptotic effect of chemotherapy. Of the various downstream targets of AKT, we examined activation of the NF-κB transcription factor and subsequent transcriptional regulation of BCL-2 gene family in pancreatic cancer cells. Inhibition of either phosphatidylinositol-3 kinase or AKT led to a decreased protein level of the antiapoptotic gene BCL-2 and an increased protein level of the proapoptotic gene BAX. Furthermore, inhibition of AKT decreased the function of NF-κB, which is capable of transcriptional regulation of the BCL-2 gene. Inhibiting this pathway had little effect on the basal level of apoptosis in pancreatic cancer cells, but increased the apoptotic effect of chemotherapy. The antiapoptotic effect of AKT activation in pancreatic cancer cells may involve transcriptional induction of a profile of BCL-2 proteins that confer resistance to apoptosis; alteration of this balance allows sensitisation to the apoptotic effect of chemotherapy.

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.

Schedule-dependent molecular effects of the proteasome inhibitor bortezomib and gemcitabine in pancreatic cancer.

BACKGROUND 26S proteasome inhibitors are a novel class of compounds entering clinical trials as a method to increase tumor sensitivity to standard chemotherapy. We determined the effect of alternate sequencing regimens of a proteasome inhibitor and gemcitabine on molecular and cellular responses in pancreatic cancer cells. MATERIALS AND METHODS MIA-PaCa-2 human pancreatic cancer cells were treated with the proteasome inhibitor bortezomib either before, simultaneously or following exposure to gemcitabine. Expression of the cell cycle proteins p21(WAF1/CIP1) and p27(KIP1), and the anti-apoptotic protein BCL-2 were determined by Western blotting. Cell cycle changes and immediate or delayed induction of apoptosis were quantitated. RESULTS Gemcitabine followed by bortezomib induced the greatest induction of apoptosis and long-term inhibition of cell growth. Bortezomib treatment led to accumulation of p21(WAF1/CIP1) and p27(KIP1) and decreased BCL-2; gemcitabine decreased p27(KIP1), induced BCL-2 and had no effect on p21(WAF1/CIP1). When these agents were given in combination or sequence, intermediate changes in these proteins were observed, and the alterations did not correlate with immediate or delayed induction of apoptosis. CONCLUSIONS Inhibition of the 26S proteasome following chemotherapy appears to be the most effective regimen, though changes in BCL-2, p21(WAF1/CIP1), p27(KIP1) do not necessarily correlate with the cellular effects when various sequences are examined. Therefore, these proteins may not be the most appropriate surrogate markers of efficacy of this regimen. These data provide the background for the development of the optimal regimen to be used in clinical trials.

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.

Sentinel Lymph Node Biopsy for Breast Cancer

Sentinel lymph node biopsy (SLNB) is an emerging surgical technique to improve lymph node staging for breast cancer. Despite the rapid development of this technique, there remain aspects of SLNB that need to be further defined to provide a standardized approach. Variables, including patient selection, technical details for the performance of SLNB, extent of pathologic evaluation of the sentinel lymph node, and the impact of micrometastases, are areas of controversy. This paper reviews the controversies and discusses available data as well as personal experience and opinion.