• Bi-directional regulation between RNF6 and AKT: Potential mediators of prostate cancer progression

      Shimelis, Hermela; Qiu, Yun (2013)
      Currently androgen deprivation therapy (ADT) is the initial treatment for men with metastatic prostate cancer. Despite the initial response to ADT, nearly all men develop advanced castrate-resistant prostate cancer (CRPC). Due to the lack of effective therapy for CRPC patients, the biology of such tumors has been under intense investigation. Previously, our laboratory demonstrated that protein expression of ring finger protein 6 (RNF6) ubiquitin ligase is increased in advanced human prostate tumors and that RNF6 was required for the growth of CRPC. The purpose of this study was to identify mechanisms by which RNF6 promote growth of CRPC cells. Using mass spectrometry, several RNF6 associated proteins were identified, one of which was AKT kinase (also called protein kinase B). A growing body of data suggests that AKT kinase promotes development and progression of CRPC. This study identifies novel roles the RNF6 ubiquitin ligase and AKT kinase in the growth of CRPC through mechanisms involving cross-regulation between the two proteins. We demonstrate that RNF6 is required for basal and growth factor mediated AKT kinase activity. RNF6 induces AKT polyubiquitination at lysine 183 within the kinase domain and this ubiquitination is required for the kinase activity. Additionally AKT ubiquitination at lysine 183 is required for proper nuclear/cytosol distribution and disruption of association with PH domain and leucine rich repeat protein phosphatase (PHLPP), thereby maintaining AKT phosphorylation required for the kinase activity. Functional assays reveal that RNF6 induced AKT ubiquitination at lysine 183 is required for prostate cancer cells growth under androgen depleted conditions. Mass spectrometry analysis also reveals that AKT phosphorylates RNF6 at serine 219. AKT induced RNF6 phosphorylation at serine 219 is required for RNF6 association with the androgen receptor (AR) and regulation of its activity. This is a novel mechanism of AR regulation by AKT through an effector protein, RNF6. In conclusion, RNF6 regulates AKT kinase activity through ubiquitination. On the other hand, AKT mediated RNF6 phosphorylation is required for RNF6 induced upregulation of AR activity. These are novel roles of AKT and RNF6 in driving castrate resistant growth of prostate cancer cells.
    • Regulation of Class III Beta-Tubulin by Src-Mediated Tyrosine Phosphorylation

      Alfano, Alan; Qiu, Yun (2015)
      Prostate cancer (PCa) is the second most lethal cancer in men, accounting for an estimated 30,000 deaths in 2013. For diagnoses of advanced disease, the standard treatment is androgen deprivation therapy (ADT). Despite initial success of ADT, many PCa patients relapse into an incurable androgen-insensitive disease termed Castration Resistant Prostate Cancer (CRPC). Taxane compounds (such as docetaxel [DTX]) + Steroid (such as Prednisone) is a first-line treatment, but DTX resistance is common in patients with advanced CRPC. Tubulins are an integral part of the cytoskeleton, and play a pivotal role in cell signaling, migration, and division. They exist in vivo either as soluble monomers or as α-/β-tubulin heterodimers. These dimers are either recycled into monomers, or polymerized into microtubules (MTs). Microtubules serve a range of functions including structural support of the cell body, scaffolding for signaling molecules, and mitotic spindle support. β-tubulin is also the molecular target for taxane compounds. High expression levels of Class III β-tubulin (TUBB3, a primarily neural isoform of β-tubulin) correlate with taxane resistance and poor prognosis in several human cancers, including ovarian cancer (serous adenocarcinoma), breast cancer, non-small-cell lung cancer (NSCLC), and prostate cancer (PCa). It is known that c-Src (a known proto-oncogene) phosphorylates β-tubulins during both hematopoietic and neural differentiation. This has also been observed in leukemia cells. Despite recent advances, the relationship between Src-mediated tyrosine phosphorylation and microtubule modulation is still poorly understood, especially in the context of advanced solid malignancies. Herein we show that activated Src-kinase is able to phosphorylate TUBB3 at tyrosine 340 (Y340), and that tyrosine phosphorylation of βIII-tubulin at Y340 by Src-family kinases is critical in stabilization of TUBB3, and also plays a role in regulation of mitotic spindles. Given the clinical utility of TUBB3 as a biomarker of poor prognosis, characterizing cancer-specific post-translational modification (PTM) of TUBB3 in aggressive cancer cell types could lead to more specified biomarkers of patient outcome or therapeutic response. Additionally, a deeper understanding of the complex interplay between the Src family of kinases (SFKs) and microtubules could enable future researchers to design more specific and effective microtubule-targeted and/or SFK-targeted therapies.
    • The role of Pim-1 kinases in advanced prostate cancer therapeutic resistance

      Linn, Douglas Ernest; Qiu, Yun (2011)
      The standard of care for patients with advanced prostate cancer is hormone ablation, a treatment that is often initially effective, but eventually fails. Second line therapy options are limited and patients frequently acquire resistance to the chemotherapeutic drugs used. Pim-1 kinases have previously been described as diagnostic biomarkers for prostate cancer progression and serve roles in chemoresistance. Here we investigated the role of Pim-1 kinases during advanced prostate cancer progression focusing on how they may promote resistance and subsequent failure of both hormone ablation and chemotherapy approaches. The androgen receptor (AR) has a central role in castration resistance, a stage where many prostate cancer patients exhibit recurrent tumor growth even under androgen ablation. Our studies demonstrate that Pim-1 kinases can regulate AR activity in different ways. The 33 kDa isoform Pim-1S induces AR degradation, a process which has been previously shown to be a critical process for cell cycle progression of prostate cancer cells. The 44 kDa isoform Pim-1L stabilizes AR levels thereby enhancing AR-mediated transcription and expression of downstream target genes. Both Pim-1 kinase isoforms were able to promote prostate cancer cell growth under androgen depleted conditions, which is a potential contributing factor that may lead to castration resistance. Characterization of drug-resistant prostate cancer cell lines revealed that Pim-1L and pluripotency transcription factor OCT4 were upregulated. We observed transcriptionally active OCT4 was critical for the enhanced tumorigenicity of these resistant lines, as knock-down by short hairpin RNA dramatically decreased in vitro and in vivo tumor growth. Pim-1 target sites T235/S236 were found to be essential for target gene expression and transcriptional activity. Together these experiments suggested Pim-1 kinases could promote maintenance of an aggressive tumor-initiating cell population through OCT4 signaling and provided yet another mechanism by which Pim-1 kinases could facilitate therapeutic failure. In summary, we have provided evidence that Pim-1 kinases can promote resistance to prostate cancer therapy by two interrelated mechanisms. Our data suggest that targeting Pim-1 kinases may prove valuable in preventing therapy resistance or for resensitizing patients to chemotherapeutic drugs currently used for treatment.

      Deshmukh, Dhanraj; Qiu, Yun (2017-)
      Prostate cancer is one of the most commonly diagnosed cancers in men worldwide. Ubiquitin E3 ligases play an important role in carcinogenesis by modulating the expression levels of important cell cycle checkpoint proteins. The CDK2 inhibitor p27 (Kip1) is an important regulator of the G1/S checkpoint. Reduced nuclear p27 levels have previously been shown to be an independent predictor of decreased time between prostatectomy and biochemical recurrence. Here we investigated the role of the E3 ubiquitin ligase RNF6 in cell cycle progression in prostate cancer. Our data demonstrate that RNF6 can promote cell cycle progression by modulating the levels of p27. Knockdown of RNF6 led to an increase in the stability of p27 and to the arrest of cells in the G1 phase. RNF6 interacted with p27 via its KIL domain and this interaction was found to be phosphorylation independent. RNF6 enhanced the ubiquitination and subsequent degradation of p27 in the early G0/G1 phase of the cell cycle. Knockdown of RNF6 by short hairpin RNA led to the inhibition of the CDK2/Cyclin E complex thereby reducing the phosphorylation of the retinoblastoma protein (pRb) and to subsequent decrease in cell cycle progression and proliferation. In addition to its role in cell cycle regulation, RNF6 is also involved in the modulation of PARP-1 activity. Both PARP-1 and CDK2 play an important role in DNA repair. Since RNF6 regulates the activities of both, CDK2 and PARP-1, we hypothesized that combining a PARP-1/2 inhibitor Veliparib to a CDK2 inhibitor Dinaciclib might sensitize prostate cancer cells to the CDK2 inhibitor and enhance cytotoxicity. Treatment of LNCaP and CWR-R1 cells with Dinaciclib at pharmacologically relevant dose levels, led to a significant induction of apoptosis. Simultaneously treating DU145 cells with a PARP-1 inhibitor, Veliparib, led to enhanced PARP-1 cleavage which is consistent with apoptosis. In summary, we have provided evidence that p27 is a substrate for RNF6. Dinaciclib alone or in combination with Veliparib could be used as a therapeutic intervention in prostate cancer.
    • Targeting the crosstalk between AR3 and E2F1 as a prospective therapy for drug-resistant prostate cancer

      Xu, Jin; Qiu, Yun (2020)
      Background: Drug resistance is one of the most prevalent causes of death in advanced prostate cancer patients. Combination therapies that target cancer cells via different mechanisms to overcome resistance have gained increased attention in recent years. However, the optimal drug combinations and the underlying mechanisms are yet to be fully explored. Aim and methods: The aim of this study is to investigate drugs that inhibit the growth of cells that are resistant to standard chemo and androgen deprivation therapy, and determine the underlying mechanisms of their action. To achieve this aim, we established cell lines that are resistant to this standard combination drug treatment and tested new compounds to overcome this “double drug” resistance. Results: Our results show that combination of enzalutamide (ENZ) and docetaxel (DTX) effectively inhibit the growth of prostate cancer cells that are resistant to either DTX or ENZ alone. The downregulation of transcription factor E2F1 plays a crucial role in cellular inhibition in response to the combined therapy. Notably, the androgen receptor (AR) variant AR3 (a.k.a. AR-V7), but not AR full length (AR-FL), positively regulates E2F1 expression in these cells. Specifically, E2F1 regulates AR3 and forms a positive regulatory feed-forward loop. Moreover, this drug combination treatment also results in DNA double strand break via the E2F1-AR3 signaling axis. Importantly, we established new drug-resistant cell lines that are resistant to ENZ+DTX combination therapy and found that the expression of both AR3 and E2F1 was restored in these double drug-resistant cells. Furthermore, we identified that auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, overcame the drug resistance and inhibited the growth of drug-resistant prostate cancer cells both in vitro and in vivo. Conclusion and significance: This proof-of-principle study demonstrates that targeting the E2F1/AR3 feedforward loop via a combination therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer.