Abstract
Growth factor-induced activation of protein kinase-B (PKB), also known as AKT, induces pro-survival signaling and inhibits activation of pro-apoptotic signaling molecules including the Forkhead box O-3a (FOXO3a) transcription factor and caspase in transformed prostate cells in vitro. Earlier we reported that Withaferin-A (WA), a small herbal molecule, induces pro-apoptotic response-4 (Par-4) mediated apoptosis in castration-resistant prostate cancer (CRPC) cells. In the present study, we demonstrate that inhibition of AKT facilitates nuclear shuttling of FOXO3a where it regulates Par-4 transcription in CRPC cells. FOXO3a is upstream of Par-4 signaling, which is required for induction of apoptosis in CRPC cells. Promoter bashing studies and Ch-IP analysis confirm a direct interaction of FOXO3a and Par-4; a sequential deletion of FOXO3a-binding sites in the Par-4 promoter fails to induce Par-4 activation. To confirm these observations, we either overexpressed AKT or silenced FOXO3a activation in CRPC cells. Both methods inhibit Par-4 function and apoptosis is significantly compromised. In xenograft tumors derived from AKT-overexpressed CRPC cells, FOXO3a and Par-4 expression is downregulated, leading to aggressive tumor growth. Oral administration of WA to mice with xenograft tumors restores FOXO3a-mediated Par-4 functions and results in inhibited tumor growth. Finally, an inverse correlation of nuclear localization of AKT expression corresponds to cytoplasmic Par-4 localization in human prostate tissue array. Our studies suggest that Par-4 is one of the key transcriptional targets of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a appears to be an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC.
Highlights
FOXO3a has a pivotal role in both oncogenesis and tumor suppression.[9]
These results reveal that WA inhibits AKT activity and induces Prostate apoptosis response-4 (Par-4), which correlates with WA-induced cytotoxicity in castration-resistant prostate cancer (CRPC) cells
We demonstrate that AKT activation inhibits the pro-apoptotic function of Par-4 by suppressing FOXO3a activation in CRPC cells
Summary
FOXO3a has a pivotal role in both oncogenesis and tumor suppression.[9]. Loss of FOXO3a has been observed in various cancers, and its cellular localization and phosphorylation status are considered to be prognostic factors for breast,[10] prostate,[11,12] bladder,[13] and ovarian cancer.[14]. FOXO3a triggers cell-cycle arrest and apoptosis through expression of genes necessary for cell death,[15] including Fas ligand (FasL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and Bim.[16] Overexpression of FOXO proteins induces apoptosis in cells of various tissue types and possesses tumor-suppressor functions.[17] Together, these studies suggest that FOXO3a functions as a tumor suppressor, and may serve as either a direct or indirect target for cancer therapy.[18]. Prostate apoptosis response-4 (Par-4), known as PAWR, is a tumor-suppressor protein that is reported to cause apoptosis in cancer cells[19,20] by activating both intrinsic and extrinsic pathways.[21] Par-4 is expressed in diverse normal and cancerous cell types and tissues, and resides in both the cytoplasm and the nucleus. Some patients eventually become refractory to such treatments, having developed what is known as castration-resistant prostate cancer (CRPC).[28]
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