Shazli N. Malik

Phosphorylation of Akt (Ser473) is an Excellent Predictor of Poor Clinical Outcome in Prostate Cancer

We previously showed, by immunohistochemistry with phospho-specific antibodies, increased phosphorylation (activation) of Akt (Ser473) [phosphorylated Akt (pAkt)] in high-Gleason grade prostate cancer (Malik SN, et al., Clin Cancer Res 2002;8:1168–71). Elevation of pAkt was accompanied by decreased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) [phosphorylated ERK (pERK)], indicative of inactivation. In this report, we determined whether increased pAkt and decreased pERK predicted clinical outcome. Prostate-specific antigen (PSA) failure (detectable and rising PSA) versus PSA non-failure (undetectable PSA 5 years after prostatectomy) was used as a surrogate for clinical outcome. Prostate tumors from cases of PSA failure versus non-failure were stained for pAkt and pERK. A significant increase in mean pAkt staining (P < 0.001) in the PSA failures versus non-failures was seen based on the Wilcoxon signed ranks test [222.18 ± 33.9 (n = 37) versus 108.79 ± 104.57 (n = 16)]. Using the best-fitting multiple logistic regression equation, a 100-point increase in pAkt staining resulted in a 160% increase in the odds of being a PSA failure. There was decreased staining for pERK in PSA failures versus non-failures: a 100-point decrease resulted in an 80% increase in the odds of being a PSA failure. Each of these effects assumed the other biomarker was held constant. The area under the receiver-operating characteristic curve for these two biomarkers predicting PSA failure was 0.84, indicating excellent discrimination between PSA failure and non-failure cases. These data indicate that increased pAkt, alone or together with decreased pERK, is an important predictor of probability of PSA failure. However, pERK alone was not a significant predictor of PSA failure.

Determining Risk of Biochemical Recurrence in Prostate Cancer by Immunohistochemical Detection of PTEN Expression and Akt Activation

Purpose: A considerable fraction of patients who undergo radical prostatectomy as treatment for primary prostate cancer experience biochemical recurrence detected by elevated serum levels of prostate-specific antigen. In this study, we investigate whether loss of expression of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the phosphorylated form of the cell survival protein Akt (pAkt) predicts biochemical recurrence. Experimental Design: Expression of PTEN and pAkt was detected by immunohistochemistry in paraffin-embedded prostate cancer tissue obtained from men undergoing radical prostatectomy. Outcome was determined by 60-month follow-up determining serum prostate-specific antigen levels. Results: By itself, PTEN was not a good predictor of biochemical recurrence; however, in combination with pAkt, it was a better predictor of the risk of biochemical recurrence compared with pAkt alone. Ninety percent of all cases with high pAkt and negative PTEN were recurrent whereas 88.2% of those with low pAkt and positive PTEN were nonrecurrent. In addition, high Gleason scores resulted in reduced protection from decreased pAkt and increased PTEN. By univariate logistic regression, pAkt alone gives an area under the receiver-operator characteristic curve of 0.82 whereas the area under the receiver-operator characteristic curve for the combination of PTEN, pAkt, and Gleason based on a stepwise selection model is 0.89, indicating excellent discrimination. Conclusions: Our results indicate that loss of PTEN expression, together with increased Akt phosphorylation and Gleason score, is of significant predictive value for determining, at the time of prostatectomy, the risk of biochemical recurrence.

Extracellular domain of TGFβ type III receptor inhibits angiogenesis and tumor growth in human cancer cells

TGFβ overexpression in human cancer cells has been shown to promote tumor progression. In the present study, we sought to determine whether sequestration of endogenous TGFβ by the expression of a soluble TGFβ type III receptor (sRIII), can reduce malignancy in human carcinoma cells and whether the tumor-suppressive activity of sRIII is associated with the inhibition of angiogenesis. Ectopic expression of sRIII significantly inhibited the growth of tumors formed by human colon carcinoma HCT116 and breast carcinoma MDA-MB-435 cells in nude mice. It also reduced the metastatic potential of the MDA-MB-435 cells. Thus, endogenous TGFβ appears to be necessary for the progression of these two carcinomas. Furthermore, when the tumor cells were mixed with Matrigel and embedded subcutaneously in nude mice, the blood volume in Matrigel plugs containing sRIII-expressing cells as indicated by hemoglobin levels was significantly lower than that in Matrigel plugs containing the respective control cells. Blood vessel counts in paraffin sections of the Matrigel plugs containing sRIII-expressing cells were also significantly lower than those in paraffin sections of the Matrigel plugs containing control cells. Treatment of human endothelial cells with a recombinant sRIII significantly inhibited their ability to form a capillary web structure on Matrigel. These results for the first time indicate that the sRIII-induced tumor suppression appears to be in part due to the inhibition of angiogenesis.

Akt in Prostate Cancer: Possible Role in Androgen-Independence

Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), has often been implicated in prostate cancer. Studies in prostate tumor cell lines revealed that Akt activation is probably important for the progression of prostate cancer to an androgen-independent state. Investigations of human prostate cancer tissues show that although there is neither Akt gene amplification nor enhanced protein expression in prostate cancer compared to normal tissue, poorly differentiated tumors exhibit increased expression of a phosphorylated (activated) form of Akt compared to normal tissue, prostatic intraepithelial neoplasia (PIN) or well-differentiated prostate cancer. Akt phosphorylation is accompanied by the inactivation of ERK, a member of the mitogen activated protein kinase (MAPK) family. In this article, we postulate that Akt promotes androgen-independent survival of prostate tumor cells by modulating the expression and activation of the androgen receptor (AR).

A Phase I Pharmacokinetic and Biological Correlative Study of Oblimersen Sodium (Genasense, G3139), an Antisense Oligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients with Hormone-Refractory Prostate Cancer

Purpose: To assess the feasibility of administering oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the Bcl-2 mRNA, with docetaxel to patients with hormone-refractory prostate cancer; to characterize the pertinent pharmacokinetic parameters, Bcl-2 protein inhibition in peripheral blood mononuclear cell(s) (PBMC) and tumor; and to seek preliminary evidence of antitumor activity. Experimental Design: Patients were treated with increasing doses of oblimersen sodium administered by continuous i.v. infusion on days 1 to 6 and docetaxel administered i.v. over 1 h on day 6 every 3 weeks. Plasma was sampled to characterize the pharmacokinetic parameters of both oblimersen and docetaxel, and Bcl-2 protein expression was measured from paired collections of PBMCs pretreatment and post-treatment. Results: Twenty patients received 124 courses of the oblimersen and docetaxel combination at doses ranging from 5 to 7 mg/kg/day oblimersen and 60 to 100 mg/m2 docetaxel. The rate of severe fatigue accompanied by severe neutropenia was unacceptably high at doses exceeding 7 mg/kg/day oblimersen and 75 mg/m2 docetaxel. Nausea, vomiting, and fever were common, but rarely severe. Oblimersen mean steady-state concentrations were 3.44 ± 1.31 and 5.32 ± 2.34 at the 5- and 7-mg/kg dose levels, respectively. Prostate-specific antigen responses were observed in 7 of 12 taxane-naïve patients, but in taxane-refractory patients no responses were observed. Preliminary evaluation of Bcl-2 expression in diagnostic tumor specimens was not predictive of response to this therapy. Conclusions: The recommended Phase II doses for oblimersen and docetaxel on this schedule are 7 mg/kg/day continuous i.v. infusion days 1 to 6, and 75 mg/m2 i.v. day 6, respectively, once every 3 weeks. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition in PBMC and tumor tissue, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination.