BIRC6 (Baculoviral IAP repeat-containing 6)

The BIRC6 gene (BRUCE/APOLLON) encodes the cytoplasmic protein BIRC6 in mammals, consisting of a single N-terminal baculoviral IAP repeat (BIR) domain and a C-terminal ubiquitin-conjugating (UBC) domain. Of the huge protein size at 528 kDa, BIRC6 demonstrated pleiotropic functions including inhibition of apoptosis, cytoprotection, regulation of cytokinesis, mitosis, autophagy and neutrophil differentiation. With the BIR domain, BIRC6 is defined as a member of the Inhibitor of Apoptosis (IAP) family. Through its BIR domain, BIRC6 binds to active caspases, including caspases-3, 6, 7 and 9 and accounts for its ability to inhibit the caspase cascade and ultimately apoptosis. The UBC domain has chimeric E2/E3 ubiquitin ligase activity where it facilitates proteosomal degradation of various proteins, including pro-apoptotic proteins p53, caspases, Smac and mitotic regulator cyclin A. More importantly, the UBC domain plays an indispensable role in embryonic development in mammals and spermatogenesis in Drosophila. Increasing evidence supports the cancer promoting role of BIRC6. Elevated BIRC6 expression has been found in a variety of cancers and was shown to contribute to treatment resistance

BIRC6 as novel therapeutic target in advanced prostate cancer : clinical relevance, development of potential therapeutic agents & preclinical drug efficacy

The lack of effective therapy for advanced prostate cancer (PCa) remains a major unmet clinical need. Recently approved therapeutics, such as enzalutamide (ENZ), have only delayed the inevitable progression of castration-resistant PCa (CRPC), as resistance will typically emerge following treatment. Although increased apoptosis-resisting ability of cancer cells represents a fundamental mechanism for the onset of treatment resistance, no relevant agents have yet been developed. Preliminary work in our laboratory has revealed an association between elevated expression of BIRC6, an Inhibitor of Apoptosis (IAP) protein, and advanced PCa. The overall objective of this doctoral study is to investigate the roles of BIRC6 in advanced PCa, and to assess the therapeutic efficacy of a novel anti-BIRC6 agent. Firstly, I evaluated the clinical relevance of BIRC6 using patients’ PCa specimens, and the functional importance of BIRC6 using cell line-based PCa models. A significant correlation was found between elevated BIRC6 protein expression in clinical PCa and poor patient prognostic factors. Functional assays validated the importance of BIRC6 in PCa cell proliferation and apoptosis suppression. Next, I designed BIRC6-based, dual IAP-targeting antisense oligonucleotides (dASOs) to inhibit BIRC6 and an additional IAP. Two dASOs, 6w2 and 6w5 targeting BIRC6+cIAP1 and BIRC6+survivin, showed substantial inhibition of CRPC cell proliferation in vitro and in vivo. Functional studies showed that both dASOs significantly induced apoptosis, cell cycle arrest and suppression of NFκB activation in CRPC cells. Finally, I assessed the growth-inhibitory efficacy of dASO-6w2 in ENZ-resistant CRPC, which has become an increasingly prominent problem in the clinic. The efficacy of dASO-6w2 was studied using both ENZ-resistant PCa cell lines and a clinically relevant, transplantable patient-derived xenograft PCa tissue model, designated LTL-313BR, which exhibits primary ENZ resistance. Importantly, I showed that treatment with dASO-6w2 markedly suppressed the growth of LTL-313BR xenografts. The dASO-6w2 was also found to increase tumour apoptosis and inhibit the expression of several pro-survival genes that were up-regulated in the LTL-313BR line. In conclusion, this doctoral study has established the clinical relevance and functional importance of BIRC6 in advanced PCa, and has also presented new BIRC6-targeting agents that markedly suppress the growth of advanced PCa.Medicine, Faculty ofGraduat

The potential effect of bioactive food supplements in targeting prostate cancer stem cells

published_or_final_versionAnatomyMasterMaster of Philosoph

Effects of PSP on CSC properties.

<p>A) Spheroid formation assay was performed with PC-3 and Du145 cells. Two hundred of cells were seeded onto polyHEMA pre-coated plates and treated with either 500 µg/mL of PSP or vehicle for 14 days. The number of prostaspheres formed was counted, and the result was presented as the mean ± s.d. Note that γ-T3 treatment efficiently suppresses the spheroid formation ability of PC-3 cells. Image of the prostaspheres was captured under microscope. Note that no prostaspheres can be found in cells treated with 500 µg/mL of PSP. (B) PSP inhibited the formation of secondary prostaspheres. Primary prostaspheres were dissociated and re-seeded into polyHEMA pre-coated plate. PSP was added 24 hr after the plating. Note that prostasphere formation was inhibited by more than 70% and 90% in the presence of 250 µg/mL and 500 µg/mL of PSP respectively. * <i>P</i><0.001, ** <i>P</i><0.0001, <i>t</i> test.</p

PSP down-regulates prostate CSC markers in PC-3 cells.

<p>A) Western blotting of prostate CSC markers CD44 and CD133 in PC-3 cells after PSP treatment. Note that PSP significantly down-regulates both stem cell markers in a dose- and time-dependent manner. B) Viability of PC-3 cells after treatment with 5, 25, 125, 250 and 500 µg/ml of PSP for 48 or 72 hr was measured with MTT assay. Results are presented as mean ± s.d. C) Flow cytometry analysis of PC-3 cells after treatment with 250 µg/ml of PSP for 72 hr. Note that no significant difference in cell cycle distribution was observed. D) Western blotting results for apoptotic markers (left panel) and stem cell maintenance proteins (right panel) in PC-3 cells after PSP treatment. Note that no changes in Bax and Bcl-2 or cleavage of PARP were detected.</p

Effect of PSP on PIN development in the TgMAP transgenic mouse model.

<p>A) Time frame of the PIN and PCa development in TgMAP mice and the schedule of the PSP treatment. Fourteen-week old TgMAP mice were treated with 200 mg/kg of PSP by oral gavage feeding for 4 weeks and sacrificed at the time when PIN has developed (20 weeks old). The table summarizes the results of the histology examination of the prostate from the vehicle- and PSP-treated TgMAP mice. C) Representative photos of the Hematoxylin & Eosin staining of the prostatic tissues from the TgMAP mice. Note that both control- and PSP-treated TgMAP mice developed prostatic intraepithelial neoplasia (PIN), as indicated by the arrows.</p

Effect of PSP on prostate tumor development of the TgMAP transgenic mouse model.

<p>A) Outline of the schedule for PSP treatment. Eight-week old TgMAP mice were treated with 300 mg/kg of PSP by oral gavage feeding for 20 weeks and sacrificed at age of 28 weeks. B & C) Representative photos of the Hematoxylin & Eosin staining of the prostate tissues from the vehicle and PSP-treated TgMAP mice. Note that tumors were found in all of the mice that were treated with vehicle only but were absent in all the PSP-treated mice. D) The table summarizes the results of the histology examination of the prostate tissues from the vehicle and PSP-treated TgMAP mice. *P<0.05 compared to control treatment by Fisher's exact test. E) Average body weight of the mice during the PSP treatment.</p

Correction: Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer