Co-targeting SKP2 and KDM5B inhibits prostate cancer progression by abrogating AKT signaling with induction of senescence and apoptosis.

BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.

Bortezomib in Combination with Physachenolide C Reduces the Tumorigenic Properties of KRASmut/P53mut Lung Cancer Cells by Inhibiting c-FLIP.

BACKGROUND: Defects in apoptosis regulation are one of the classical features of cancer cells, often associated with more aggressiveness and failure to therapeutic options. We investigated the combinatorial antitumor effects of a natural product, physachenolide C (PCC) and bortezomib, in KRASmut/P53mut lung cancer cells and xenograft mice models. METHODS: The in vitro anticancer effects of the bortezomib and PCC combination were investigated using cell viability, migration, and invasion assays in 344SQ, H23, and H358 cell lines. Furthermore, the effects of combination treatment on the critical parameters of cellular metabolism, including extracellular acidification rate (ECAR) and mitochondrial oxidative phosphorylation based on the oxygen consumption rate of cancer cells were assessed using Seahorse assay. Finally, the antitumor effect of the bortezomib (1 mg/kg) and PCC (10 mg/kg) combination was evaluated using xenograft mice models. RESULTS: Our data showed that the bortezomib-PCC combination was more effective in reducing the viability of lung cancer cells in comparison with the individual treatments. Similarly, the combination treatment showed a significant inhibition of cell migration and invasion of cancer cells. Additionally, the key anti-apoptotic protein c-FLIP was significantly inhibited along with a substantial reduction in the key parameters of cellular metabolism in cancer cells. Notably, the bortezomib or PCC inhibited the tumor growth compared to the control group, the tumor growth inhibition was much more effective when bortezomib was combined with PCC in tumor xenograft mice models. CONCLUSION: These findings demonstrate that PCC sensitizes cancer cells to bortezomib, potentially improving the antitumor effects against KRASmut/P53mut lung cancer cells, with an enhanced efficacy of combination treatments without causing significant side effects.

Lysosome-dependent FOXA1 ubiquitination contributes to luminal lineage of advanced prostate cancer.

Changes in FOXA1 (forkhead box protein A1) protein levels are well associated with prostate cancer (PCa) progression. Unfortunately, direct targeting of FOXA1 in progressive PCa remains challenging due to variations in FOXA1 protein levels, increased FOXA1 mutations at different stages of PCa, and elusive post-translational FOXA1 regulating mechanisms. Here, we show that SKP2 (S-phase kinase-associated protein 2) catalyzes K6- and K29-linked polyubiquitination of FOXA1 for lysosomal-dependent degradation. Our data indicate increased SKP2:FOXA1 protein ratios in stage IV human PCa compared to stages I-III, together with a strong inverse correlation (r = -0.9659) between SKP2 and FOXA1 levels, suggesting that SKP2-FOXA1 protein interactions play a significant role in PCa progression. Prostate tumors of Pten/Trp53 mice displayed increased Skp2-Foxa1-Pcna signaling and colocalization, whereas disruption of the Skp2-Foxa1 interplay in Pten/Trp53/Skp2 triple-null mice demonstrated decreased Pcna levels and increased expression of Foxa1 and luminal positive cells. Treatment of xenograft mice with the SKP2 inhibitor SZL P1-41 decreased tumor proliferation, SKP2:FOXA1 ratios, and colocalization. Thus, our results highlight the significance of the SKP2-FOXA1 interplay on the luminal lineage in PCa and the potential of therapeutically targeting FOXA1 through SKP2 to improve PCa control.

Improving combination therapies: targeting A2B-adenosine receptor to modulate metabolic tumor microenvironment and immunosuppression.

BACKGROUND: We investigated the role of A2B-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A2B-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. METHODS: The antitumor efficacy of A2B-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO2, pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. RESULTS: Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A2B-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon γ production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval = 3.35 to 41.13], n = 10, P < .001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. CONCLUSIONS: Data identify A2B-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

MicroRNA-21 deficiency suppresses prostate cancer progression through downregulation of the IRS1-SREBP-1 signaling pathway.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy.

Small-Molecule Natural Product Physachenolide C Potentiates Immunotherapy Efficacy by Targeting BET Proteins.

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. SIGNIFICANCE: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Regulation of cell cycle by MDM2 in prostate cancer cells through Aurora Kinase-B and p21WAF1/CIP1 mediated pathways.

Overexpression of MDM2 oncoprotein has been detected in a large number of diverse human malignancies and has been shown to play both p53-dependent and p53-independent roles in oncogenesis. Our study was designed to explore the impact of MDM2 overexpression on the levels of various cell cycle regulatory proteins including Aurora kinase-B (AURK-B), CDC25C and CDK1, which are known to promote tumor progression and increase metastatic potential. Our data from human cell cycle RT2 profiler PCR array experiments revealed significant changes in the expression profile of genes that are involved in different phases of cell cycle regulation in LNCaP-MST (MDM2 transfected) prostate cancer cells. Our current study has demonstrated a significant increase in the expression level of AURK-B, CDC25C, Cyclin A2, Cyclin B and CDK1 in LNCaP-MST cells as compared with wild type LNCaP cells that were modulated by MDM2 specific inhibitor Nutlin-3. In fact, the expression levels of the above- mentioned proteins were significantly altered at both mRNA and protein levels after treating the cells with 20 µM Nutlin-3 for 24h. Additionally, the pro-apoptotic proteins including p53, p21, and Bax were elevated with the concomitant decrease in the key anti-apoptotic proteins following MDM2 inhibitor treatment. Also, Nutlin-3 treated cells demonstrated caspase-3 activation was observed with an in-vitro caspase-3 fluorescent assay performed with caspase 3/7 specific DEVD-amc substrate. Our results offer significant evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest via blocking the transmission of signals through AURKB-CDK1 axis and inducing apoptosis in LNCaP-MST cancer cells. It is evident from our data that MDM2 overexpression probably is the primary cause for CDK1 up-regulation in the LNCaP-MST cells, which might have occurred possibly through activation of AURK-B. However, further studies in this direction should shed more light on the intracellular mechanisms involved in the regulation of Aurora kinase-B and CDK1 axis in MDM2 positive cancers.

MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells.

The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT² profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.

Anti-angiogenic and pro-apoptotic effects of a small-molecule JFD-WS in in vitro and breast cancer xenograft mouse models.

A small molecule that was developed for blocking vascular endothelial growth factor receptor 2 (VEGFR2) has been tested and confirmed for its anti-angiogenic activity. Subsequently, it was modified into a water soluble salt form (JFD-WS) to increase bioavailability and distribution during in vivo pre-clinical testing. The present study was designed to further evaluate the anti-angiogenic and pro-apoptotic effects of JFD-WS in monotherapy as well as in combination with paclitaxel (Taxol) using a mouse xenograft model. The in vitro anti-angiogenic effects of JFD-WS were investigated using cell proliferation, migration, Matrigel tube formation and VEGFR2 phosphorylation assays. The anti-angiogenic effect of JFD-WS was further established using chorioallantoic membrane (CAM) assay followed by in vivo efficacy testing on GI-101A breast adenocarcinoma cells. Pharmacokinetic and toxicity studies were performed using BALB/c mice. Finally, the apoptotic signals were assessed in the control and experimental tumor samples, and the plasma mucin 1 (MUC1) levels were analyzed. In the in vitro tests, JFD-WS effectively inhibited HUVEC proliferation, migration, tube formation and VEGFR2 phosphorylation. Additionally, JFD-WS inhibited the formation of blood vessels in chick chorioallantoic membrane. While inhibiting the xenograft tumor growth in experimental mice, JFD-WS decreased the plasma MUC1 levels. The western blot analysis of apoptotic markers and fragmentation analysis of DNA confirmed the pro-apoptotic effects of JFD-WS. These results indicated that JFD-WS alone or in combination with paclitaxel exerted antitumor and pro-apoptotic effects in the breast cancer xenograft model due to an anti-angiogenic effect. These results strongly support the clinical translation of its use.

Anti-cancer effects of F16: A novel vascular endothelial growth factor receptor-specific inhibitor.

Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancers. This study was aimed to explore the anti-angiogenic activity of a novel vascular endothelial growth factor receptor-specific inhibitor named F16 in both in vitro and in vivo experimental models. This compound effectively reduced cell proliferation, tube formation, and migration of human umbilical vein endothelial cells in a concentration-dependent manner by directly inhibiting vascular endothelial growth factor binding and subsequent vascular endothelial growth factor receptor-2 phosphorylation. The F16 was also able to inhibit the phosphoinositide 3-kinase/protein kinase B-mediated survival and migration pathways in cancer in addition to inhibiting the focal adhesion kinase and mitogen-activated protein kinases-mediated signaling in GI-101A cancer cells. The chorioallantoic membrane assay followed by tumor growth inhibition measurements with GI-101A breast cancer xenograft implanted athymic nude mice confirmed the in vivo tumor reductive effects of F16. It was interesting to observe a decrease in tumor burden after F16 treatment which correlated very well with the decrease in the plasma levels of mucin-1 (MUC-1). Our studies so far have confirmed that F16 is a specific inhibitor of angiogenesis in both in vitro and in vivo models. The F16 also works very efficiently with Taxol in combination by limiting the tumor growth that is better than the monotherapy with any one of the drugs that were tested individually. Thus, F16 offers a promising anti-proliferative and anti-angiogenic effects with better specificity than some of the existing multi-kinase inhibitors.