Kang-Sup Shim

Biochemical Characterization of the Human RAD51 Protein III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES

Adenosine nucleotides affect the ability of RecA·single-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPγS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPγS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51·ssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA·ssDNA binding form. The available evidence suggests that a low molecular weight hRAD51·ssDNA binding form (hRAD51·ssDNAlow) correlates with active ADP and ATP processing. A high molecular weight hRAD51·ssDNA aggregate (hRAD51·ssDNAhigh) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

Biochemical Characterization of the Human RAD51 Protein II. ADENOSINE NUCLEOTIDE BINDING AND COMPETITION

RecA mediated homologous recombination requires cooperative ATP binding and hydrolysis to assume and maintain an active, extended DNA-protein (nucleoprotein) filament. Human RAD51 protein (hRAD51) lacks the magnitude of ATP-induced cooperativity and catalytic efficiency displayed by RecA. Here, we examined hRAD51 binding and ATPase inhibition pattern by ADP and ATP/adenosine 5′-O-(thiotriphosphate) (ATPγS). hRAD51 fully saturates with ATP/ATPγS regardless of DNA cofactor (K D ≈ 5 μm; 1 ATP/1 hRAD51). The binding of ADP to hRAD51 appeared bimodal. The first mode was identical to ATP/ATPγS binding (K app1 ≈ 3 μm; 1 ADP/1 hRAD51), while a second mode occurred at elevated ADP concentrations (K app2 ≥ 125 μm; >1 ADP/1 hRAD51). We could detect ADP → ATP exchange in the high affinity ADP binding mode (K app1) but not the low affinity binding mode (K app2). At low ATP concentrations (<0.3 mm), ADP and ATPγS competitively inhibit the hRAD51 ATPase (K m (app) >K m ). However, at high ATP (>0.3 mm), the hRAD51 ATPase was stimulated by concentrations of ATPγS that were 20-fold above the K D . Ammonium sulfate plus spermidine decreased the affinity of hRAD51 for ADP substantially (∼10-fold) and ATP modestly (∼3-fold). Our results suggest that ATP binding is not rate-limiting but that the inability to sustain an active nucleoprotein filament probably restricts the hRAD51 ATPase.

hMSH4-hMSH5 Adenosine Nucleotide Processing and Interactions with Homologous Recombination Machinery

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437–451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP→ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.

Noncatalytic PTEN missense mutation predisposes to organ-selective cancer development in vivo

Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.

Crosstalk between PKCα and PI3K/AKT Signaling Is Tumor Suppressive in the Endometrium

SUMMARY Protein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis.

Abstract 189: Protein kinase C alpha suppresses AKT activation in endometrial cancer

Endometrial cancer (EC) is the most common gynecological malignancy and the fourth leading cancer in women in the United States. Hyperactivation of the PI3K/AKT pathway is observed in ∼90% of EC cases and this pathway is the most prominent driver of endometrial carcinogenesis. Mutations in multiple PI3K/AKT pathway components (including PTEN, PIK3CA, PIK3R1, AKT) often coexist in EC, indicating that EC development requires full, unopposed activation of PI3K/AKT signaling. In this study, we identified PKCα as a negative regulator of AKT activation that suppresses endometrial carcinogenesis. Our analysis revealed that PKCα protein and mRNA are reduced or lost in 50% of EC cell lines and ∼60% of human ECs. PKCα deficiency was also observed in hyperplastic endometrial lesions in murine models with allelic knock-in of mutant Pten (ptenΔ4-5/+, ptenC124R/+ and ptenC129E/+) or endometrial-specific Pten deletion (ptenpr-/-, ptenltf-/-), indicating that loss of PKCα is an early event in endometrial carcinogenesis. Mechanistic analysis has further revealed that loss of PKCα is independent of changes in PI3K/AKT signaling; however, the enzyme acts as a potent negative regulator of the PI3K/AKT pathway in EC cells. PKC agonists reduced AKT phosphorylation/activity in EC cells that retain PKCα, via a PP2A-dependent mechanism. Conversely, PKCα knockdown led to increased AKT phosphorylation. Notably, while PKC agonists did not affect AKT in EC cells with loss of PKCα, exogenous expression of the enzyme in these cells restored the inhibitory effect of the agonists on PI3K/AKT signaling. Collectively these data indicate that PKCα has a restraining effect on PI3K/AKT signaling in EC cells, providing a basis for its loss during endometrial tumor progression. Physiological relevance of these findings was established using cell lines, patient samples and mouse models. PKCα restoration in human EC cells decreased expression of oncogenes such as cyclin D1 and Id1 and abolished their anchorage-independent growth, supporting a tumor suppressive role for the isozyme in this tissue. Analysis of PKCα expression in patient specimens determined that loss of PKCα expression correlates with high grade disease. Finally, in mice heterozygous for mutant Pten, PKCα knockout led to a 3-fold increase in endometrial tumor burden at three months, confirming a role of PKCα as a tumor suppressor in the endometrium. Taken together, our results provide evidence that PKCα 1) negatively regulates AKT signaling and the expression of important oncogenes in EC cells; and 2) functions as tumor suppressor in the endometrium whose loss is associated with disease progression. Thus, PKCα signaling represents a promising biomarker for risk stratification in early stage disease and may provide insight into therapeutic strategies for late stage disease. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojiang Chen, Subodh M. Lele, Takiko Daikoku, Sudhansu K. Dey, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha suppresses AKT activation in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 189.

Abstract 4720: Protein kinase C alpha (PKCα) regulates PI3K/AKT signaling in endometrial cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer in women in the United States. Although loss of PTEN and deregulation of PI3K/AKT signaling has been implicated in approximately 90% of endometrial cancer cases, understanding of the molecular etiology of the disease remains limited. Our analysis of tissue from 448 endometrial cancer patients has determined that ∼60% of human tumors show reduced expression or loss of the signal transduction molecule PKCα. Loss of this enzyme is also seen in endometrial hyperplasias arising in mouse models carrying germline mutations of PTEN (PtenΔ4-5, PtenC124R and PtenG129E) or endometrial specific deletion of PTEN (Ptenpr-/−), indicating that loss of PKCα can be an early event in development of the disease. In patients, reduced expression of PKCα correlates with markers of disease aggressiveness, such as increased myometrial invasion and lymph node involvement, supporting a tumor suppressive role for the enzyme in the endometrium. This correlation is particularly marked in endometrioid, PTEN-mutant disease, pointing to a potential link between PKCα and PI3K/AKT signaling. The role of PKCα in endometrial cancer was further examined using a panel of 17 human endometrial cancer cell lines with varying expression of the kinase. Restoration of PKCα in cell lines lacking the enzyme blocked their ability to grow in soft agarose, further supporting a tumor suppressive role of the kinase in this tissue. Stimulation of cells that express PKCα with PKC agonists reduced phosphorylation/activation of AKT and promoted loss of downstream PI3K/AKT targets such as cyclin D1 and inhibitor of DNA binding 1 (Id1). Analysis of the PKC isozyme expression profile in these cells combined with the use of selective inhibitors pointed to PKCα as the mediator of the effect. The demonstration that PKC agonists failed to inhibit AKT signaling in cell lines that lack expression of PKCα, and that expression of exogenous PKCα restored the effect, confirmed the involvement of PKCα. The inhibition of AKT by PKCα is dependent on PP2A since okadaic acid and calyculin A, but not the PHLPP1/2 inhibitors NSC117079 and NSC45586, blocked the effect, a finding consistent with the ability of PKCα to inhibit AKT activity in PTEN mutant cell lines. Taken together, our study provides evidence that 1) loss of PKCα is a common and early event in endometrial cancer; 2) PKCα plays a crucial role in regulating AKT activation and growth promoting signaling molecules in endometrial cancer, and 3) effects of PKCα on AKT in endometrial cancer are mediated by PP2A. Given the importance of PI3K/AKT in the disease, these findings highlight the potential of PKCα signaling as a potential biomarker for disease risk and as a potential therapeutic target in endometrial cancer. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl Morrison, Baojing Chen, Subodh M. Lele, Takiko Daikoku, Sudhansu K. Dey, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) regulates PI3K/AKT signaling in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4720. doi:10.1158/1538-7445.AM2015-4720

Abstract 4211: Protein kinase C alpha (PKCα) signaling in endometrial cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Endometrial cancer is the most common gynecological malignancy in the US and the fourth most common cancer in women. Despite its high incidence, there is limited knowledge of the molecular etiology of this disease. With obesity being a major risk factor, the incidence of endometrial cancer has been on a steady incline and is expected to become an increasing cause of cancer mortality in future years. Thus, improved understanding of endometrial tumorigenesis is of critical importance. Our analysis of human endometrial tumors identified loss of the signaling molecule PKCα in approximately 60% of endometrioid (Type I) tumors. Loss of PKCα trends with increasing tumor grade and is associated with aggressive disease features such as lymphovascular involvement and myometrial invasion, pointing to an important role for this molecule in regulating endometrial cancer progression. Inactivation of the tumor suppressor PTEN and deregulation of the PI3K/AKT pathway are key drivers of Type I endometrial cancer. Thus, mice with allele-specific knock-in of cancer-related Pten mutants (i.e., PtenΔ4-5, PtenC124R, and PtenG120E) offer unique models for the disease. Precancerous endometrial hyperplasias arising in these mice show loss of PTEN and increased AKT activity. While PKCα is expressed in the normal murine endometrial epithelium at all phases of the estrus cycle, the enzyme is uniformly lost in these precancerous lesions. Thus, disruption of PKCα signaling can occur early in endometrial tumorigenesis. In human endometrial cancer cell lines, low PKCα levels also correlate with PTEN loss; however, shRNA-mediated knockdown of PTEN in PTEN expressing cells did not affect PKCα expression indicating that downregulation of the kinase is not the direct result of changes in PTEN activity. Notably, PKC agonists suppress AKT activity in cells expressing high levels PKCα (HEC-1-A, HEC-50) but not in PKCα-low cells (Ishikawa, RL95-2). Collectively, these data suggest that, in addition to PTEN inactivation, loss of PKCα is required for full activation of the PI3K/AKT signaling pathway during endometrial carcinogenesis. Analysis of the molecular basis for loss of PKCα expression in endometrial tumors identified multiple regulatory mechanisms. PKCα protein expression in tumors generally parallels that of its mRNA and promoter activity assays point to transcriptional mechanisms for PKCα downregulation. Differential mRNA and protein stability were identified as additional levels of regulation of PKCα expression in endometrial cancer. Taken together, our findings indicate that 1) PKCα signaling regulates the PI3K/AKT pathway in the endometrial epithelium; 2) concomitant loss of PKCα and PTEN may act in a co-operative manner in regulation of endometrial turmorigenesis; and 3) loss of PKCα can occur by multiple mechanisms, as often seen with tumor suppressive molecules during neoplastic progression. Supported by NIH grants CA036727, CA016056, and DK60632. Citation Format: Alice H. Hsu, Kathryn J. Curry, Kang-Sup Shim, Peter Frederick, Carl D. Morrison, Baojing Chen, Subodh M. Lele, Gustavo Leone, Adrian R. Black, Jennifer D. Black. Protein kinase C alpha (PKCα) signaling in endometrial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2014-4211