Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells

Cifuentes, Eugenia; Mataraza, Jennifer; Yoshida, Barbara A.; Menon, Mani; Sacks, David B.; Barrack, Evelyn R.; Reddy, G. Prem Veer

doi:10.1073/pnas.0307161101

Cited by 41 publications

(60 citation statements)

References 47 publications

(46 reference statements)

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“…5B). During the second scenario, NTAP-AR could bind CaM through a direct physical interaction with AR as noted previously (45). Regrettably the results cannot discern either scenario.…”

Section: Table I Summary Of Proteomics Datacontrasting

confidence: 39%

Identification of Putative Androgen Receptor Interaction Protein Modules

Jasavala

Martinez

Thumar

et al. 2007

Molecular & Cellular Proteomics

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We have developed a novel androgen receptor (AR) expression system in the 293 human embryonic kidney cell line that recapitulates AR biochemical activity as a steroid hormone receptor in prostate cancer cells. We used this system to identify putative AR-binding proteins in the cytosolic and nuclear compartments of mammalian cells using a large scale co-immunoprecipitation strategy coupled to quantitative mass spectrometry. For example, the heat shock 70 and 90 chaperones, which are known regulators of steroid hormone receptor, were identified as AR-binding proteins. AR purification enriched for proteins involved in RNA processing, protein transport, and cytoskeletal organization, suggesting a functional link between AR and these protein modules in mammalian cells. For example, AR purification in the nuclear compartment led to the specific enrichment of ␣-actinin-4, clathrin heavy chain, and serine-threonine protein kinase C ␦. Short interfering RNA knockdown studies and co-transcriptional reporter assays revealed that clathrin heavy chain possessed co-activator activity during AR-mediated transcription, whereas ␣-actinin-4 and protein kinase C ␦ displayed both co-activator and co-repressor activity during AR-mediated transcription that was dependent upon their relative expression levels. Lastly immunohistochemical staining of prostate tissue showed that ␣-actinin-4 levels decreased in the nucleus of high grade cancerous prostate samples, suggesting its possible deregulation in advanced prostate cancers as previously observed in late stage metastatic breast cancers. Taken together, these findings suggest AR binds to specific protein modules in mammalian cells and that these

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“…5B). During the second scenario, NTAP-AR could bind CaM through a direct physical interaction with AR as noted previously (45). Regrettably the results cannot discern either scenario.…”

Section: Table I Summary Of Proteomics Datacontrasting

confidence: 39%

Identification of Putative Androgen Receptor Interaction Protein Modules

Jasavala

Martinez

Thumar

et al. 2007

Molecular & Cellular Proteomics

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“…More recently, an association with the signaling protein calmodulin has been also shown in LNCaP cells. 35 In the case of src, the interaction is due to a proline-rich region in the AR, 14 whereas in the case of caveolin-1 both the NH 2 -terminal and ligand-binding domains of the AR are involved. 15 Both src and caveolin-1 are capable of interacting with EGFR and are involved EGF-stimulated signaling.…”

Section: Egfr Internalization Is Altered In Pc3-ar Cellsmentioning

confidence: 99%

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

Bonaccorsi

Carloni

Muratori

et al. 2004

Intl Journal of Cancer

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We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of ␣6␤4 integrin expression. The treatment with androgens further reduced invasion of the cells without modifying ␣6␤4 expression, suggesting an interference with the invasion process by androgens. Here, we investigated EGF-mediated signal transduction processes that lead to invasion in PC3-AR cells. We show that EGF-induced EGFR autotransphosphorylation is reduced in PC3-AR cells compared to PC3 cells transfected only with the vector (PC3-Neo). EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, and EGF-PI3K interaction are also decreased in PC3 Key words: prostate cancer; epidermal growth factor receptor; androgen receptor; PI3K; invasionProstate Cancer (PC) is one of the most common cancer and the second leading cause of death in American men. 1 Since prostate cancer cell growth is enhanced by androgens, in the advanced stages of the disease, androgen ablation therapy represents a valuable tool for the treatment of these patients. However the development in most patients after few years of treatment of androgenindependent clones, characterized by higher invasiveness and metastatic properties, has focused attention on the molecular mechanisms that lead to loss of androgen-dependence as well as on the pathways that are regulated by androgens in these cells besides proliferation. Indeed, although androgens are the major stimulus for proliferation of prostate cancer cells, maintenance of androgensensitivity appears to keep a more differentiated and less malignant phenotype of these cells. The ability to produce tumors in nude mice, for instance, is higher in androgen-insensitive cell lines (such as PC3 and DU145) with respect to androgen sensitive (LNCaP). 2 In this light, the role of androgens in the regulation of the pathways involved in invasion and metastasis represents a major task in studies on prostate cancer biology. 3 As a result, some androgen-regulated genes involved in signaling pathways that lead to invasion have been recently identified 4 -6 and their role in decreasing invasion ability of androgen-sensitive prostate cancer cells indicated. Migration and invasion of cancer cells is regulated by multiple pathways that employ various growth factor and their receptors, integrins and cytoskeletal elements. A key role is played by the EGF receptor (EGFR), which, following interaction with the integrin ␣6␤4, promotes cell migration through activation of PI3K and other downstream pathways. 7,8 In a previous study, we demonstrated that the expression of androgen receptor in PC3 cells by transfection with a full-length human androgen receptor expression vector (PC3-AR) determined a decrease in the expression of the integrin ␣6␤4 and in the ability of these cells to invade Matrigel in response to EGF. 6 The treatment with the synthetic androgen R1881 determined a ...

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“…Whole-cell extracts were prepared essentially as described by Cifuentes et al (28). Briefly, LNCaP cells were suspended at a density of 2 Â 10 7 /mL in buffer A and sonicated and the extracts were clarified as above.…”

Section: Methodsmentioning

confidence: 99%

“…The activity of the DNA-bound complex is governed by binding of coactivator and corepressor proteins to the LBD and the ATD. Finally, AR activity is also controlled by AR-calmodulin (CaM) interaction (28). It is the effect of AR interaction with CaM on the breakdown of AR that we examine herein.…”

Section: Introductionmentioning

confidence: 99%

Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

Pelley¹,

Chinnakannu²,

Murthy³

et al. 2006

Cancer Research

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Chemotherapy of prostate cancer targets androgen receptor (AR) by androgen ablation or antiandrogens, but unfortunately, it is not curative. Our attack on prostate cancer envisions the proteolytic elimination of AR, which requires a fuller understanding of AR turnover. We showed previously that calmodulin (CaM) binds to AR with important consequences for AR stability and function. To examine the involvement of Ca 2+ /CaM in the proteolytic breakdown of AR, we analyzed LNCaP cell extracts that bind to a CaM affinity column for the presence of low molecular weight forms of AR (intact AR size, f114 kDa). Using an antibody directed against the NH 2 -terminal domain (ATD) of AR on Western blots, we identified f76-kDa, f50-kDa, and 34/31-kDa polypeptides in eluates of CaM affinity columns, suggesting the presence of CaM-binding sites within the 31/34-kDa ATD of AR. Under cell-free conditions in the presence of phenylmethylsulfonyl fluoride, AR underwent Ca 2+ -dependent degradation. AR degradation was inhibited by N-acetyl-leu-leu-norleu, an inhibitor of thiol proteases, suggesting the involvement of calpain. In intact cells, AR breakdown was accelerated by raising intracellular Ca 2+ using calcimycin, and increased AR breakdown was reversed with the cell-permeable Ca 2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N ¶,N ¶-tetraacetic acid tetra-(acetoxymethyl)-ester. In CaM affinity chromatography studies, the Ca 2+ -dependent protease calpain was bound to and eluted from the CaM-agarose column along with AR. Caspase-3, which plays a role in AR turnover under stress conditions, did not bind to the CaM column and was present in the proenzyme form. Similarly, AR immunoprecipitates prepared from wholecell extracts of exponentially growing LNCaP cells contained both calpain and calpastatin. Nuclear levels of calpain and calpastatin (its endogenous inhibitor) changed in a reciprocal fashion as synchronized LNCaP cells progressed from G 1 to S phase. These reciprocal changes correlated with changes in AR level, which increased in late G 1 phase and decreased as S phase progressed. Taken together, these observations suggest potential involvement of AR-bound CaM in calciumcontrolled, calpain-mediated breakdown of AR in prostate cancer cells. (Cancer Res 2006; 66(24): 11754-62)

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Physical and functional interaction of androgen receptor with calmodulin in prostate cancer cells

Cited by 41 publications

References 47 publications

Identification of Putative Androgen Receptor Interaction Protein Modules

Identification of Putative Androgen Receptor Interaction Protein Modules

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells

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