ABSTRACT. 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.

SummaryLow-dose exposures to common environmental chemicals that are deemed safe individually may be combining to instigate carcinogenesis, thereby contributing to the incidence of cancer. This risk may be overlooked by current regulatory practices and needs to be vigorously investigated.

Filamin A (FlnA) has been associated with actin as cytoskeleton regulator. Recently its role in the cell has come under scrutiny for FlnA’s involvement in cancer development. FlnA was originally revealed as a cancer-promoting protein, involved in invasion and metastasis. However, recent studies have also found that under certain conditions, it prevented tumor formation or progression, confusing the precise function of FlnA in cancer development. Here, we try to decipher the role of FlnA in cancer and the implications for its dual role. We propose that differences in subcellular localization of FlnA dictate its role in cancer development. In the cytoplasm, FlnA functions in various growth signaling pathways, such as vascular endothelial growth factor, in addition to being involved in cell migration and adhesion pathways, such as R-Ras and integrin signaling. Involvement in these pathways and various others has shown a correlation between high cytoplasmic FlnA levels and invasive cancers. However, an active cleaved form of FlnA can localize to the nucleus rather than the cytoplasm and its interaction with transcription factors has been linked to a decrease in invasiveness of cancers. Therefore, overexpression of FlnA has a tumor-promoting effect, only when it is localized to the cytoplasm, whereas if FlnA undergoes proteolysis and the resulting C-terminal fragment localizes to the nucleus, it acts to suppress tumor growth and inhibit metastasis. Development of drugs to target FlnA and cause cleavage and subsequent localization to the nucleus could be a new and potent field of research in treating cancer.

Purpose: We previously showed that nuclear localization of the actin-binding protein, filamin A (FlnA), corresponded to hormone-dependence in prostate cancer. Intact FlnA (280 kDa, cytoplasmic) cleaved to a 90 kDa fragment which translocated to the nucleus in hormone-naI« ve cells, whereas in hormone-refractory cells, FlnA was phosphorylated, preventing its cleavage and nuclear translocation. We have examined whether FlnA localization determines a propensity to metastasis in advanced androgen-independent prostate cancer. Experimental Design: We examined, by immunohistochemistry, FlnA localization in paraffinembedded human prostate tissue representing different stages of progression. Results were correlated with in vitro studies in a cell model of prostate cancer. Results: Nuclear FlnA was significantly higher in benign prostate (0.6612 F 0.5888), prostatic intraepithelial neoplasia (PIN; 0.6024 F 0.4620), and clinically localized cancers (0.69134 F 0.5686) compared with metastatic prostate cancers (0.3719 F 0.4992, P = 0.0007). Cytoplasmic FlnA increased from benign prostate (0.0833 F 0.2677), PIN (0.1409 F 0.2293), localized cancers (0.3008 F 0.3762, P = 0.0150), to metastases (0.7632 F 0.4414, P < 0.00001). Logistic regression of metastatic versus nonmetastatic tissue yielded the area under the receiver operating curve as 0.67 for nuclear-FlnA, 0.79 for cytoplasmic-FlnA, and 0.82 for both, indicating that metastasis correlates with cytoplasmic to nuclear translocation. In vitro studies showed that cytoplasmic localization of FlnA induced cell invasion whereas nuclear translocation of the protein inhibited it. FlnA dephosphorylation with the protein kinaseA inhibitor H-89 facilitated FlnA nuclear translocation, resulting in decreased invasiveness and AR transcriptional activity, and induced sensitivity to androgen withdrawal in hormone-refractory cells. Conclusions:The data presented in this study indicate that in prostate cancer, metastasis correlates with cytoplasmic localization of FlnA and may be prevented by cleavage and subsequent nuclear translocation of this protein.Filamins are a family of cytoskeletal proteins that organize filamentous actin into networks and stress fibers (1). Filamin A (FlnA) is a 280 kDa non -muscle actin binding protein, the appropriate function of which is essential for development (2, 3). FlnA dimerization forms a V-shaped flexible structure which can induce high-angle orthogonal branching and efficiently gather actin filaments into a three-dimensional gel in vitro by cross-linking actin filaments at the leading edge of migrating cells. Hence, filamins are essential for mammalian cell locomotion, anchoring of transmembrane proteins including integrins, and also act as interfaces for protein-protein interaction (4). More than 30 proteins of great functional diversity are known to interact with filamins which function as a signaling scaffold by connecting and coordinating a large variety of cellular processes (4).In prostate cancer, a role for FlnA was identified in prost...

The structure of the entire chmmel-fonning bacte1iocin (protein toxin) colicin Iahas been solved to a resolution of2.4A by multiple isomorphous replacement. The fonnation of ion-penneable chmmels in ta.rget cell membrm1es is a general mechanism of cytotoxicity. The process involves secretion of a soluble protein which inserts into the plasma membrm1e of the target cell m1d fon11S a lethal pore. Colicins, Escherichia coli protein toxins, are a well-characterized exmnple of this class of proteins. Colicin Ia crystals, comprised of approximately 80% solvent are in spacegroup C222 1 (a=64.4A, b=l78.6A, c=285.5A). All data sets used in the structure determination were collected fi·om fi·ozen crystals with a synchrotr·on light source (SSRL bean1line 7-1 ). Heavy atom de1ivatives were obtained using mercmial soaks of engineered single-site cysteine mutants.The str1.1cture of the 69kD colicin Ia protein reveals the structural relationships between the three distinct domains which function, respectively, to i) bind to a receptor on the outer membrane of susceptible bacte1ia, ii) tr·anslocate across the outer membrane tlu·ough the receptor, and iii) bind to the inner membrane and fom1 a pore in the presence of the transmembrane voltage. The domains are sepm·ated by an extraordinmily long helical coiled-coil. The cqstal structure of the a-;sembly domain of the cmtilage oligomeric matrix protein (COMP), a pentameric glycoprotein of the thrombospondin family found in cmtilage m1d tendon, was detem1ined at 2.03 A resolution using MIRAS phasing with xenon, (CH3)3Pb(COOCH3)3 and Pr(COOCH3)3 fmther improved by solvent flattenincr and five~fold avera£ing. Self-association ofCOMP, as well as of at le~t two other extracellul~ matrix proteins, thrombosponilins 3 and 4, is achieved through the fonmtion of a five-str·m1ded a-helical bundle which involves 64 N-tem1inal residues (20-83). The complex is fiuther stabilized by the interchain disulphide bonds between cysteines 68 m1d 71. Circular dichroism measurements show that the structme of the assembly domain remains intact even at temperatures above I OOOC. Wlllie the cryst£'11 structures of two-, tlu·ee-and fom-stranded a-helical bm1dles were reported before, that one of tl1e pentai11eric coiled coil is novel. The migins of the extreme thennal stability, the unusual degree of oligomelization and tl1e role of the intemal hydrophobic axial cavity are tl1e questions to be addressed in tl1e cmTent study. The peptides containing 64, 52 or 46 residues were produced by expression in Escherichia coli, but well diffracting crystals were obt£1ined only witl1 t11e 46 residues fragment (Ph a=38.47 A, b=49.47 A, c= 54.98 A and ~= 103.84°). The centr·al pmt of tl1e molecule w11ich includes five heptad repeats (residues 29-65), obeys approximate five-fold symmetry while the remaining residues at theN-and C-tem1ini show significant deviations fi·om tl1at. Str·ong sy1m11etry violations could explain tl1e little success acl1ieved in om earlier attempts to solve t11e structure by tl1e molecuhu·replacem...

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with antiandrogen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD þ FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgenreduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

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.

Prostate cancer is initially dependent on androgens for growth; hence, recurrent prostate is treated with androgen ablation which may result in progression to androgen independence characterized by a resistance to such therapy. Androgens bind to and activate the androgen receptor (AR), a member of the nuclear steroid receptor family of transcription factors, which regulates prostate cancer cell proliferation and survival in androgen-independent, as well as -dependent, tumors. Another pathway regulating proliferation and survival is the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Here we analyze reports in the literature indicating that these two pathways cooperate to regulate prostate tumor development and progression. Studies show that AR transcriptional activity and expression are regulated by Akt. In addition, androgens regulate the Akt pathway by both genomic and non-genomic effects. This explains why prostate tumors subjected to androgen ablation experience an increase in Akt phosphorylation, and suggest that the tumor compensates for the loss of one pathway with another. Different modes of interaction between the two pathways, including direct interaction, or regulation via downstream intermediates, such as the wnt/GSK-3beta/beta-catenin pathway, NF-kappaB, and the FOXO family of transcription factors, will be discussed. In addition, we will discuss the role of Akt in the interaction of the AR with upstream regulators of Akt phosphorylation, such as receptor tyrosine kinases of the EGF and IGF-1 receptor families and the tumor suppressor PTEN.