Houjian Cai

Cell Autonomous Role of PTEN in Regulating Castration-Resistant Prostate Cancer Growth

Alteration of the PTEN/PI3K pathway is associated with late-stage and castrate-resistant prostate cancer (CRPC). However, how PTEN loss is involved in CRPC development is not clear. Here, we show that castration-resistant growth is an intrinsic property of Pten null prostate cancer (CaP) cells, independent of cancer development stage. PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of Ar in the epithelium promotes the proliferation of Pten null cancer cells, at least in part, by downregulating the androgen-responsive gene Fkbp5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy.

Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression

Dominant mutations or DNA amplification of tyrosine kinases are rare among the oncogenic alterations implicated in prostate cancer. We demonstrate that castration-resistant prostate cancer (CRPC) in men exhibits increased tyrosine phosphorylation, raising the question of whether enhanced tyrosine kinase activity is observed in prostate cancer in the absence of specific tyrosine kinase mutation or DNA amplification. We generated a mouse model of prostate cancer progression using commonly perturbed non-tyrosine kinase oncogenes and pathways and detected a significant up-regulation of tyrosine phosphorylation at the carcinoma stage. Phosphotyrosine peptide enrichment and quantitative mass spectrometry identified oncogene-specific tyrosine kinase signatures, including activation of EGFR, ephrin type-A receptor 2 (EPHA2), and JAK2. Kinase:substrate relationship analysis of the phosphopeptides also revealed ABL1 and SRC tyrosine kinase activation. The observation of elevated tyrosine kinase signaling in advanced prostate cancer and identification of specific tyrosine kinase pathways from genetically defined tumor models point to unique therapeutic approaches using tyrosine kinase inhibitors for advanced prostate cancer.

Invasive Prostate Carcinoma Driven by c-Src and Androgen Receptor Synergy

Cellular Src (c-Src) integrates a large number of signal transduction pathways regulating cell division, migration, and other aspects of cell physiology. Mutations of Src kinase have not been described in human prostate cancer, but evidence for increased levels of expression accompanying cancer progression has been reported. We analyzed overexpression of c-Src in naïve mouse prostate epithelium and observed no change in tubule formation frequency or histologic structure. However, when enhanced c-Src expression is coupled with enhanced expression of androgen receptor (AR), it results in a strong activation of Src kinase activity accompanied by activation of the MAPK pathway, and enhanced AR activity. Similar to the pathology induced by constitutively active c-Src(Y529F), the tubules progress to frank carcinoma with invasion and display markers of epithelial-to-mesenchymal transition. These combined results suggest that nonmutated Src kinase may play a more important role in the genesis and progression of prostate cancer than previously appreciated and that epigenetic changes that enhance the level of AR may select for enhanced expression of c-Src with accompanying activation and a strong drive to malignant progression.

Differential transformation capacity of Src family kinases during the initiation of prostate cancer

Src family kinases (SFKs) are pleiotropic activators that are responsible for integrating signal transduction for multiple receptors that regulate cellular proliferation, invasion, and metastasis in a variety of human cancers. Independent groups have identified increased expression of individual SFK members during prostate cancer progression, raising the question of whether SFKs display functional equivalence. Here, we show that Src kinase, followed by Fyn kinase and then Lyn kinase, exhibit ranked tumorigenic potential during both paracrine-induced and cell-autonomous–initiated prostate cancer. This quantitative variation in transformation potential appears to be regulated in part by posttranslational palmitoylation. Our data indicate that development of inhibitors against specific SFK members could provide unique targeted therapeutic strategies.

Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via β-catenin signaling

The cell surface protein Trop2 is expressed on immature stem/progenitor-like cells and is overexpressed in many epithelial cancers. However the biological function of Trop2 in tissue maintenance and tumorigenesis remains unclear. In this study, we demonstrate that Trop2 is a regulator of self-renewal, proliferation, and transformation. Trop2 controls these processes through a mechanism of regulated intramembrane proteolysis that leads to cleavage of Trop2, creating two products: the extracellular domain and the intracellular domain. The intracellular domain of Trop2 is released from the membrane and accumulates in the nucleus. Heightened expression of the Trop2 intracellular domain promotes stem/progenitor self-renewal through signaling via β-catenin and is sufficient to initiate precursor lesions to prostate cancer in vivo. Importantly, we demonstrate that loss of β-catenin or Trop2 loss-of-function cleavage mutants abrogates Trop2-driven self-renewal and hyperplasia in the prostate. These findings suggest that heightened expression of Trop2 is selected for in epithelial cancers to enhance the stem-like properties of self-renewal and proliferation. Defining the mechanism of Trop2 function in self-renewal and transformation is essential to identify new therapeutic strategies to block Trop2 activation in cancer.

Differential Regulation and Substrate Preferences in Two Peptide Transporters of Saccharomyces cerevisiae

ABSTRACT Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [14C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [14C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.

Role of autonomous androgen receptor signaling in prostate cancer initiation is dichotomous and depends on the oncogenic signal

The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronic-activated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.

Development of a Method for the Determination of Acyl-CoA Compounds by Liquid Chromatography Mass Spectrometry to Probe the Metabolism of Fatty Acids

Acyl-Coenzyme As (acyl-CoAs) are a group of activated fatty acid molecules participating in multiple cellular processes including lipid synthesis, oxidative metabolism of fatty acids to produce ATP, transcriptional regulation, and protein post-translational modification. Quantification of cellular acyl-CoAs is challenging due to their instability in aqueous solutions and lack of blank matrices. Here we demonstrate an LC-MS/MS analytical method which allows for absolute quantitation with broad coverage of cellular acyl-CoAs. This assay was applied to profile endogenous acyl-CoAs under the challenge of a variety of dietary fatty acids in prostate and hepatic cells. Additionally, this approach allowed for detection of multiple fatty acid metabolic processes including the biogenesis of acyl-CoAs, and their elongation, degradation, and desaturation. Hierarchical clustering in the remodeling of acyl-CoA profiles revealed a fatty-acid-specific pattern across all tested cell lines, which provides a valuable reference for making predictions in other cell models. Individual acyl-CoAs were identified which were altered differentially by exogenous fatty acids in divergent tumorigenicity states of cells. These findings demonstrate the power of acyl-CoA profiling toward understanding the mechanisms for the progression of tumors or other diseases in response to fatty acids.

Ceramide mediates FasL-induced caspase 8 activation in colon carcinoma cells to enhance FasL-induced cytotoxicity by tumor-specific cytotoxic T lymphocytes

FasL-mediated cytotoxicity is one of the mechanisms that CTLs use to kill tumor cells. However, human colon carcinoma often deregulates the Fas signaling pathway to evade host cancer immune surveillance. We aimed at testing the hypothesis that novel ceramide analogs effectively modulate Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis. We used rational design and synthesized twenty ceramide analogs as Fas function modulators. Five ceramide analogs, IG4, IG7, IG14, IG17, and IG19, exhibit low toxicity and potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. Functional deficiency of Fas limits both FasL and ceramide analogs in the induction of apoptosis. Ceramide enhances FasL-induced activation of the MAPK, NF-κB, and caspase 8 despite induction of potent tumor cell death. Finally, a sublethal dose of several ceramide analogs significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed five novel ceramide analogs that act at a sublethal dose to enhance the efficacy of tumor-specific CTLs, and these ceramide analogs hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.

Pim1 kinase regulates c-Kit gene translation

BackgroundReceptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown.Methods and resultsWe demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1−/− mice and Pim1−/−2−/−3−/− triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1−/− and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level.ConclusionsOur study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.