Reversal of hyperactive Wnt signaling-dependent adipocyte defects by peptide boronic acids.

Deregulated Wnt signaling and altered lipid metabolism have been linked to obesity, diabetes, and various cancers, highlighting the importance of identifying inhibitors that can modulate Wnt signaling and aberrant lipid metabolism. We have established a Drosophila model with hyperactivated Wnt signaling caused by partial loss of axin, a key component of the Wnt cascade. The Axin mutant larvae are transparent and have severe adipocyte defects caused by up-regulation of ß-catenin transcriptional activities. We demonstrate pharmacologic mitigation of these phenotypes in Axin mutants by identifying bortezomib and additional peptide boronic acids. We show that the suppressive effect of peptide boronic acids on hyperactive Wnt signaling is dependent on α-catenin; the rescue effect is completely abolished with the depletion of α-catenin in adipocytes. These results indicate that rather than targeting the canonical Wnt signaling pathway directly, pharmacologic modulation of ß-catenin activity through α-catenin is a potentially attractive approach to attenuating Wnt signaling in vivo.

CDK8 mediates the dietary effects on developmental transition in Drosophila.

The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, we have previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. Here we report that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. We observed that cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on lipid metabolism and developmental timing in Drosophila larvae.

Future Aspects of CDK5 in Prostate Cancer: From Pathogenesis to Therapeutic Implications.

Cyclin-dependent kinase 5 (CDK5) is a unique member of the cyclin-dependent kinase family. CDK5 is activated by binding with its regulatory proteins, mainly p35, and its activation is essential in the development of the central nervous system (CNS) and neurodegeneration. Recently, it has been reported that CDK5 plays important roles in regulating various biological and pathological processes, including cancer progression. Concerning prostate cancer, the androgen receptor (AR) is majorly involved in tumorigenesis, while CDK5 can phosphorylate AR and promotes the proliferation of prostate cancer cells. Clinical evidence has also shown that the level of CDK5 is associated with the progression of prostate cancer. Interestingly, inhibition of CDK5 prevents prostate cancer cell growth, while drug-triggered CDK5 hyperactivation leads to apoptosis. The blocking of CDK5 activity by its small interfering RNAs (siRNA) or Roscovitine, a pan-CDK inhibitor, reduces the cellular AR protein level and triggers the death of prostate cancer cells. Thus, CDK5 plays a crucial role in the growth of prostate cancer cells, and AR regulation is one of the important pathways. In this review paper, we summarize the significant studies on CDK5-mediated regulation of prostate cancer cells. We propose that the CDK5-p35 complex might be an outstanding candidate as a diagnostic marker and potential target for prostate cancer treatment in the near future.

CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila.

The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.

Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

Regulation of lipogenic gene expression by lysine-specific histone demethylase-1 (LSD1).

Dysregulation of lipid homeostasis is a common feature of several major human diseases, including type 2 diabetes and cardiovascular disease. However, because of the complex nature of lipid metabolism, the regulatory mechanisms remain poorly defined at the molecular level. As the key transcriptional activators of lipogenic genes, such as fatty acid synthase (FAS), sterol regulatory element-binding proteins (SREBPs) play a pivotal role in stimulating lipid biosynthesis. Several studies have shown that SREBPs are regulated by the NAD(+)-dependent histone deacetylase SIRT1, which forms a complex with the lysine-specific histone demethylase LSD1. Here, we show that LSD1 plays a role in regulating SREBP1-mediated gene expression. Multiple lines of evidence suggest that LSD1 is required for SREBP1-dependent activation of the FAS promoter in mammalian cells. LSD1 knockdown decreases SREBP-1a at the transcription level. Although LSD1 affects nuclear SREBP-1 abundance indirectly through SIRT1, it is also required for SREBP1 binding to the FAS promoter. As a result, LSD1 knockdown decreases triglyceride levels in hepatocytes. Taken together, these results show that LSD1 plays a role in regulating lipogenic gene expression, suggesting LSD1 as a potential target for treating dysregulation of lipid metabolism.

Cyclin-dependent kinase 5 modulates STAT3 and androgen receptor activation through phosphorylation of Ser7²7 on STAT3 in prostate cancer cells.

Cyclin-dependent kinase 5 (Cdk5) is known to regulate prostate cancer metastasis. Our previous results indicated that Cdk5 activates androgen receptor (AR) and supports prostate cancer growth. We also found that STAT3 is a target of Cdk5 in promoting thyroid cancer cell growth, whereas STAT3 may play a role as a regulator to AR activation under cytokine control. In this study, we investigated the regulation of Cdk5 and its activator p35 on STAT3/AR signaling in prostate cancer cells. Our results show that Cdk5 biochemically interacts with STAT3 and that this interaction depends on Cdk5 activation in prostate cancer cells. The phosphorylation of STAT3 at Ser7²7 (p-Ser7²7-STAT3) is regulated by Cdk5 in cells and xenograft tumors. The mutant of STAT3 S727A reduces its interaction with Cdk5. We further show that the nuclear distribution of p-Ser7²7-STAT3 and the expression of STAT3-regulated genes (junB, c-fos, c-myc, and survivin) are regulated by Cdk5 activation. STAT3 mutant does not further decrease cell proliferation upon Cdk5 inhibition, which implies that the role of STAT3 regulated by Cdk5 correlates to cell proliferation control. Interestingly, Cdk5 may regulate the interaction between STAT3 and AR through phosphorylation of Ser7²7-STAT3 and therefore upregulate AR protein stability and transactivation. Correspondingly, clinical evidence shows that the level of p-Ser7²7-STAT3 is significantly correlated with Gleason score and the levels of upstream regulators (Cdk5 and p35) as well as downstream protein (AR). In conclusion, this study demonstrates that Cdk5 regulates STAT3 activation through Ser7²7 phosphorylation and further promotes AR activation by protein-protein interaction in prostate cancer cells.

Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5.

Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to apoptosis of prostate cancer cells. The aim of this study is to investigate how Cdk5 regulates AR activation and growth of prostate cancer cells. At first, the data show that Cdk5 enables phosphorylation of AR at Ser-81 site through direct biochemical interaction and, therefore, results in the stabilization of AR proteins. The Cdk5-dependent AR stabilization causes accumulation of AR proteins and subsequent activation. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore, decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR and contributes to prostate cancer growth. Therefore, Cdk5-p35 may be suggested as diagnostic and therapeutic targets for prostate cancer in the near future.

The significance of Her2 on androgen receptor protein stability in the transition of androgen requirement in prostate cancer cells.

Androgen ablation therapy is the most common strategy for suppressing prostate cancer progression; however, tumor cells eventually escape androgen dependence and progress to an androgen-independent phase. The androgen receptor (AR) plays a pivotal role in this transition. To address this transition mystery in prostate cancer, we established an androgen-independent prostate cancer cell line (LNCaPdcc), by long-term screening of LNCaP cells in androgen-deprived conditions, to investigate changes of molecular mechanisms before and after androgen withdrawal. We found that LNCaPdcc cells displayed a neuroendocrine morphology, less aggressive growth, and lower expression levels of cell cycle-related factors, although the cell cycle distribution was similar to parental LNCaP cells. Notably, higher protein expression of AR, phospho-Ser(81)-AR, and PSA in LNCaPdcc cells were observed. The nuclear distribution and protein stability of AR increased in LNCaPdcc cells. In addition, cell proliferation results exhibited the biphasic nature of the androgen (R1881) effect in two cell lines. On the other hand, LNCaPdcc cells expressed higher levels of Her2, phospho-Tyr(1221/1222)-Her2, ErbB3, and ErbB4 proteins than parental LNCaP cells. These two cell lines exhibited distinct responses to Her2 activation (by heregulin treatment) on Her2 phosphorylation and Her2 inhibition (by AG825 or Herceptin treatments) on proliferation. In addition, the Her2 inhibitor more effectively caused AR degradation and diminished AR Ser(81) phosphorylation in LNCaPdcc cells. Taken together, our data demonstrate that Her2 plays an important role in the support of AR protein stability in the transition of androgen requirement in prostate cancer cells. We hope these findings will provide novel insight into the treatment of hormone-refractory prostate cancer.

Involvement of cAMP in nerve growth factor-triggered p35/Cdk5 activation and differentiation in PC12 cells.

The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G(1) arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.

The role of Cdk5 in retinoic acid-induced apoptosis of cervical cancer cell line.

Cdk5 is a small serine/threonine protein kinase which belongs to Cdk family. Unlike other Cdk members, so far Cdk5 is known to be irrelevant in cell cycle. Cdk5 kinase activity is regulated by binding with its activator, p35. Our previous results indicate that CdkS and p35 are involved in drugs-induced apoptosis of prostate cancer cells. Retinoic acid (RA) is one of the vitamin A-related compounds. Because of its potency on biological functions, it has been widely studied in its novel actions including the ability to inhibit cancer cell growth and to induce apoptosis. Here, we report that RA treatment decreased the growth of human cervical cancer cell line, HeLa, and Cdk5 contributed to this effect. The involvement of Cdk5 in RA-reduced cell survival was performed by treatments of Cdk5 inhibitor and siRNA. We further identified that RA-induced growth inhibition was partly correlated to Cdk5 activity-related apoptosis by detecting cell cycle distribution of sub G1 phase and the signals of Annexin V staining. In addition, our results also indicated that Cdk5 activity was involved in RA-induced HeLa apoptosis by detecting cleavages of caspase-3 and its substrate, PARP (poly (ADP-ribose) polymerases) Interestingly, the nuclear localizations of Cdk5 and p35 proteins were increased by RA treatment, which, again, suggests the involvement of Cdk5 and p35 in RA-induced apoptotic effects. In conclusion, we provide evidence to suggest that Cdk5 and p35 might play important roles in RA-induced HeLa apoptosis.

A developmental genetic analysis of the lysine demethylase KDM2 mutations in Drosophila melanogaster.

Post-translational modification of histones plays essential roles in the transcriptional regulation of genes in eukaryotes. Methylation on basic residues of histones is regulated by histone methyltransferases and histone demethylases, and misregulation of these enzymes has been linked to a range of diseases such as cancer. Histone lysine demethylase 2 (KDM2) family proteins have been shown to either promote or suppress tumorigenesis in different human malignancies. However, the roles and regulation of KDM2 in development are poorly understood, and the exact roles of KDM2 in regulating demethylation remain controversial. Since KDM2 proteins are highly conserved in multicellular animals, we analyzed the KDM2 ortholog in Drosophila. We have observed that dKDM2 is a nuclear protein and its level fluctuates during fly development. We generated three deficiency lines that disrupt the dKdm2 locus, and together with 10 transposon insertion lines within the dKdm2 locus, we characterized the developmental defects of these alleles. The alleles of dKdm2 define three phenotypic classes, and the intragenic complementation observed among these alleles and our subsequent analyses suggest that dKDM2 is not required for viability. In addition, loss of dKDM2 appears to have rather weak effects on histone H3 lysine 36 and 4 methylation (H3K36me and H3K4me) in the third instar wandering larvae, and we observed no effect on methylation of H3K9me2, H3K27me2 and H3K27me3 in dKdm2 mutants. Taken together, these genetic, molecular and biochemical analyses suggest that dKDM2 is not required for viability of flies, indicating that dKdm2 is likely redundant with other histone lysine demethylases in regulating normal development in Drosophila.

Tumor-suppressive effects of CDK8 in endometrial cancer cells.

CDK8 is either amplified or mutated in a variety of human cancers, and CDK8 functions as an oncoprotein in melanoma and colorectal cancers. Previously, we reported that loss or reduction of CDK8 results in aberrant fat accumulation in Drosophila and mammals, suggesting that CDK8 plays an important role in inhibiting lipogenesis. Epidemiological studies have identified obesity and overweight as the major risk factors of endometrial cancer, thus we examined whether CDK8 regulates endometrial cancer cell growth by using several endometrial cancer cell lines, including KLE, which express low levels of CDK8, as well as AN3 CA and HEC-1A cells, which have high levels of endogenous CDK8. We observed that ectopic expression of CDK8 in KLE cells inhibited cell proliferation and potently blocked tumor growth in an in vivo mouse model. In addition, gain of CDK8 in KLE cells blocked cell migration and invasion in transwell, wound healing and persistence of migratory directionality assays. Conversely, we observed the opposite effects in all of the aforementioned assays when CDK8 was depleted in AN3 CA cells. Similar to AN3 CA cells, depletion of CDK8 in HEC-1A cells strongly enhanced cell migration in transwell assays, while overexpression of CDK8 in HEC-1A cells blocked cell migration. Furthermore, gene profiling of KLE cells overexpressing CDK8 revealed genes whose protein products are involved in lipid metabolism, cell cycle and cell movement pathways. Finally, depletion of CDK8 increased the expression of lipogenic genes in endometrial cancer cells. Taken together, these results show a reverse correlation between CDK8 levels and several key features of the endometrial cancer cells, including cell proliferation, migration and invasion as well as tumor formation in vivo. Therefore, in contrast to the oncogenic effects of CDK8 in melanoma and colorectal cancers, our results suggest that CDK8 plays a tumor-suppressive role in endometrial cancers.

Inhibitory effects of Rhenium-188-labeled Herceptin on prostate cancer cell growth: a possible radioimmunotherapy to prostate carcinoma.

PURPOSE: Herceptin is widely used in treating Her2-overexpressing breast cancer. However, the application of Herceptin in prostate cancer is still controversial. Our previous results have indicated the relevance of Her2 in the transition of the androgen requirement in prostate cancer cells. In this study, the effects of radioimmunotherapy against Her2 in prostate cancer were investigated. MATERIALS AND METHODS: DU145, an androgen receptor-negative prostate cancer cell line, was used in vitro and in vivo to evaluate the effects of Herceptin labeled with a beta emitter, Rhenium-188 (Re-188). Its effects on cell growth, extent of apoptosis, the bio-distribution of Re-188 labeled Herceptin (Re-H), and protein levels were determined. RESULTS: Treatments with Re-188 and Re-H reduced the proliferation of DU145 cells in dose- and time-dependent manners compared to the Herceptin-treated group. Growth inhibition and apoptosis were induced after Re-H treatment; growth inhibition was more distinct in cells with high Her2/p-Her2 levels. Our in vivo xenograft studies revealed that Re-H treatment significantly retarded tumor growth and altered the levels of apoptosis-related proteins. The bio-distribution of Re-H in mice demonstrated a tissue-specific pattern. Importantly, the levels of p35 protein, which is related to cancer cell survival and invasion, dramatically decreased after Re-H treatment. CONCLUSIONS: Our data demonstrate that Re-188-labeled Herceptin effectively inhibited the growth of DU145 cells compared to the Herceptin- and Re-188-treated cohorts. This implies that targeting Her2 by both radio- and immuno- therapy might be a potential strategy for treating patients with androgen-independent prostate cancer.