Xiaolan Guo

Dysfunctional telomeres activate an ATM‐ATR‐dependent DNA damage response to suppress tumorigenesis

The POT1 (protection of telomeres) protein binds the single‐stranded G‐rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53‐dependent growth arrest and an ATM‐dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR‐dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM‐dependent DDR. Our results uncover a novel ATR‐dependent DDR at telomeres that is normally shielded by POT1 binding to the single‐stranded G‐overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.

Multiple roles for MRE11 at uncapped telomeres.

Progressive telomere attrition or uncapping of the shelterin complex elicits a DNA damage response as a result of a cell’s inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. Telomere deprotection activates both ataxia telangiectasia mutated (ATM) and telangiectasia and Rad3-related (ATR) kinase-dependent DNA damage response pathways, and promotes efficient non-homologous end-joining (NHEJ) of dysfunctional telomeres. The mammalian MRE11–RAD50–NBS1 (MRN; NBS1 is also known as NBN) complex interacts with ATM to sense chromosomal double-strand breaks and coordinate global DNA damage responses. Although the MRN complex accumulates at dysfunctional telomeres, it is not known whether mammalian MRN promotes repair at these sites. Here we address this question by using mouse alleles that either inactivate the entire MRN complex or eliminate only the nuclease activities of MRE11 (ref. 8). We show that cells lacking MRN do not activate ATM when telomeric repeat binding factor 2 (TRF2) is removed from telomeres, and ligase 4 (LIG4)-dependent chromosome end-to-end fusions are markedly reduced. Residual chromatid fusions involve only telomeres generated by leading strand synthesis. Notably, although cells deficient for MRE11 nuclease activity efficiently activate ATM and recruit 53BP1 (also known as TP53BP1) to deprotected telomeres, the 3′ telomeric overhang persists to prevent NHEJ-mediated chromosomal fusions. Removal of shelterin proteins that protect the 3′ overhang in the setting of MRE11 nuclease deficiency restores LIG4-dependent chromosome fusions. Our data indicate a critical role for the MRN complex in sensing dysfunctional telomeres, and show that in the absence of TRF2, MRE11 nuclease activity removes the 3′ telomeric overhang to promote chromosome fusions. MRE11 can also protect newly replicated leading strand telomeres from NHEJ by promoting 5′ strand resection to generate POT1a–TPP1-bound 3′ overhangs.

Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita.

ABSTRACT The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b−/−mTR+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.

Hexokinase 2-Mediated Warburg Effect Is Required for PTEN- and p53-Deficiency-Driven Prostate Cancer Growth

Accumulating evidence suggests that codeletion of the tumor suppressor genes Pten and p53 plays a crucial role in the development of castration-resistant prostate cancer in vivo. However, the molecular mechanism underlying Pten-/p53-deficiency-driven prostate tumorigenesis remains incompletely understood. Building upon insights gained from our studies with Pten-/p53-deficient mouse embryonic fibroblasts (MEFs), we report here that hexokinase 2 (HK2) is selectively upregulated by the combined loss of Pten and p53 in prostate cancer cells. Mechanistically, Pten deletion increases HK2 mRNA translation through the activation of the AKT-mTORC1-4EBP1 axis, and p53 loss enhances HK2 mRNA stability through the inhibition of miR143 biogenesis. Genetic studies demonstrate that HK2-mediated aerobic glycolysis, known as the Warburg effect, is required for Pten-/p53-deficiency-driven tumor growth in xenograft mouse models of prostate cancer. Our findings suggest that HK2 might be a therapeutic target for prostate cancer patients carrying Pten and p53 mutations.

Methylseleninic Acid Superactivates p53-Senescence Cancer Progression Barrier in Prostate Lesions of Pten-Knockout Mouse

Monomethylated selenium (MM-Se) forms that are precursors of methylselenol, such as methylseleninic acid (MSeA), differ in metabolism and anticancer activities in preclinical cell and animal models from seleno-methionine that had failed to exert preventive efficacy against prostate cancer in North American men. Given that human prostate cancer arises from precancerous lesions such as high-grade prostatic intraepithelial neoplasia (HG-PIN), which frequently have lost phosphatase and tensin homolog (PTEN) tumor suppressor permitting phosphatidylinositol-3-OH kinase (PI3K)–protein kinase B (AKT) oncogenic signaling, we tested the efficacy of MSeA to inhibit HG-PIN progression in Pten prostate-specific knockout (KO) mice and assessed the mechanistic involvement of p53-mediated cellular senescence and of the androgen receptor (AR). We observed that short-term (4 weeks) oral MSeA treatment significantly increased expression of P53 and P21Cip1 proteins and senescence-associated-β-galactosidase staining, and reduced Ki67 cell proliferation index in Pten KO prostate epithelium. Long-term (25 weeks) MSeA administration significantly suppressed HG-PIN phenotype, tumor weight, and prevented emergence of invasive carcinoma in Pten KO mice. Mechanistically, the long-term MSeA treatment not only sustained P53-mediated senescence, but also markedly reduced AKT phosphorylation and AR abundance in the Pten KO prostate. Importantly, these cellular and molecular changes were not observed in the prostate of wild-type littermates which were similarly treated with MSeA. Because p53 signaling is likely to be intact in HG-PIN compared with advanced prostate cancer, the selective superactivation of p53-mediated senescence by MSeA suggests a new paradigm of cancer chemoprevention by strengthening a cancer progression barrier through induction of irreversible senescence with additional suppression of AR and AKT oncogenic signaling. Cancer Prev Res; 9(1); 35–42. ©2015 AACR.

Co-targeting hexokinase 2-mediated Warburg effect and ULK1-dependent autophagy suppresses tumor growth of PTEN- and TP53-deficiency-driven castration-resistant prostate cancer

Currently, no therapeutic options exist for castration-resistant prostate cancer (CRPC) patients who have developed resistance to the second generation anti-androgen receptor (AR) axis therapy. Here we report that co-deletion of Pten and p53 in murine prostate epithelium, often observed in human CRPC, leads to AR-independent CRPC and thus confers de novo resistance to second generation androgen deprivation therapy (ADT) in multiple independent yet complementary preclinical mouse models. In contrast, mechanism-driven co-targeting hexokinase 2 (HK2)-mediated Warburg effect with 2-deoxyglucose (2-DG) and ULK1-dependent autophagy with chloroquine (CQ) selectively kills cancer cells through intrinsic apoptosis to cause tumor regression in xenograft, leads to a near-complete tumor suppression and remarkably extends survival in Pten −/p53-deficiency-driven CRPC mouse model. Mechanistically, 2-DG causes AMPK phosphorylation, which in turn inhibits mTORC1-S6K1 translation signaling to preferentially block anti-apoptotic protein MCL-l synthesis to prime mitochondria-dependent apoptosis while simultaneously activates ULK1-driven autophagy for cell survival to counteract the apoptotic action of anti-Warburg effect. Accordingly, inhibition of autophagy with CQ sensitizes cancer cells to apoptosis upon 2-DG challenge. Given that 2-DG is recommended for phase II clinical trials for prostate cancer and CQ has been clinically used as an anti-malaria drug for many decades, the preclinical results from our proof-of-principle studies in vivo are imminently translatable to clinical trials to evaluate the therapeutic efficacy by the combination modality for a subset of currently incurable CRPC harboring PTEN and TP53 mutations.

Abstract 4049: The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Telomeres are nucleoprotein complexes that cap the ends of eukaryotic chromosomes and maintain chromosomal stability. Telomeres are protected by shelterin, a complex of six core proteins (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1) that prevents the chromosome ends from being mistakenly recognized as damaged DNA. Telomeres that can no longer exert end-protective functions are said to be dysfunctional. Telomere dysfunction can be caused by progressive telomere attrition (telomere shortening) or through disruption of the shelterin complex (telomere uncapping). To study whether chromosomal instability caused by telomere uncapping leads to tumorigenesis, we used the Cre-lox system to delete the telomere binding protein TRF2 in mouse embryonic fibroblasts (MEFs). In the presence of tumor suppressor protein p53, loss of TRF2 inhibited cell proliferation via induction of cellular senescence. In contrast, the absence of p53 allowed the TRF2-deficient cells to bypass senescence as these cells were negative for SA-β-galactosidase staining. However, these p53-deficient cells stopped proliferating ten days after TRF2 depletion and eventually entered telomere uncapping-induced crisis (indicated by the significant appearance of chromosomal end to end fusions, mitotic failure and induction of apoptosis). Our mechanistic studies revealed that these p53-deficient cells underwent a novel ATM-caspase 2-dependent apoptosis upon loss of TRF2. Accordingly, knocking out caspase 2 rescued these cells from apoptosis, and thus increased long-term survival of the cell colonies as well as promoted cell transformation. Mutations of p53 are predominantly missense mutations that result in accumulation of mutant p53 proteins with loss of wild-type p53 tumor-suppressive functions and gain of oncogenic activities. To study whether the gain-of-function properties of mutant p53 can rescue cells undergoing telomere crisis from cell death, we generated MEFs expressing mutant p53R172H that corresponds to the p53R175H “hot spot” mutation in human cancers. We observed that mutant p53R172H did not prevent TRF2 depletion-induced chromosomal instability as indicated by extensive chromosome fusions. However, expression of mutant p53R172H significantly rescued cells from TRF2 loss-initiated cell apoptosis and promoted cell transformation as determined by anchorage-independent soft agar assay. More importantly, mutant p53R172H promoted tumor development of TRF2-deficient MEFs in immunodeficient mice. Collectively, our findings provide compelling genetic evidence that telomere uncapping-driven chromosomal instability activates ATM-caspase 2-dependent apoptosis to suppress tumorigenesis. However, the gain-of-function properties of mutant p53 can rescue the cells undergoing telomere crisis from cell death and thus promote tumorigenesis. Citation Format: Li-Ju Chang, Fengxia Wu, Xiaolan Guo, Yibin Deng. The gain-of-function properties of mutant p53 rescues cells from telomere dysfunction-induced crisis and promotes tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4049. doi:10.1158/1538-7445.AM2013-4049

Abstract 4902: Targeting APC mutant colon cancer cells by chloroquine

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The adenomatous polyposis coli (APC) tumor suppressor is mutated in approximately 80% of sporadic colorectal cancers (CRC) where it is recognized as the critical initiating event. Genetic studies revealed that c-Myc is the crucial mediator of the early stages of tumor development following APC loss. One of the key functions of c-Myc is to repress p21 and thus switches to p53-mediated apoptosis in cancer cells. We therefore hypothesize that p21 might be an attractive target for killing APC-deficient human colon cancer cells preferentially. Here we first utilized p21-deficient human colon cancer cell line (p21−/−HCT116) to elucidate the role of p21 in colon cancer cells for apoptosis. Chloroquine (CQ), a small molecule inhibitor of autophagy, was identified to preferentially initiate p53-PUMA-dependent apoptosis in p21−/−HCT116 cells, but not in isogenic p21+/+HCT116 cells. Although CQ inhibits the late stages of autophagy by targeting lysosmoal degradation, however, the molecular basis of CQ-mediated cancer cell death remains unclear. We found that blockade of the early stage of autophagy by individual knockdown of genes involved in autophagy (Atg5, Atg7, Beclin1 and ULK1) attenuated CQ-induced accumulation of autophagosomes and apoptosis in p21−/−HCT116 cells. In addition, we showed that reactive oxygen species (ROS)-initiated autophagy was essential for CQ-mediated cancer cell death. These results suggest that inhibition of autophagy at a late stage by CQ can be detrimental to cancer cell survival when autophagy is initiated at early stage. To determine the specificity of p21 in mediating CQ-induced cancer cell death, we demonstrated that knockdown of p21 in p21+/+HCT116 cells sensitized, while ectopic expression of p21 in p21−/−HCT116 cells inhibited, CQ-induced apoptosis. Furthermore, administration of CQ in vivo significantly suppressed tumor growth in xenograft mouse model bearing the p21−/−HCT116 cells, but not the p21+/+HCT116 cells. Our data strongly support that inactivation of p21-mediated anti-apoptotic event is required for CQ to kill cancer cells and inhibit tumor growth in vivo. To examine whether what was observed in p21−/−HCT116 cells was also true for killing of APC mutant colon cancer cells due to suppression of p21 through Myc activation upon APC loss, we took shRNA-mediated knockdown approaches to effectively deplete APC in p21+/+HCT116 cells. Treatment of cancer cells with CQ specifically caused apoptosis and blocked p21 induction in APC depleted cancer cells, which correlated with a selective inhibition of tumor growth in xenograft model. Given that CQ has been safely used in patients for many years as anti-malarial and anti-rheumatoid therapies, our findings provide compelling rationale to consider clinic translational trials in colon cancer patients carrying APC mutations using lysosomotropic agents such as CQ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4902. doi:1538-7445.AM2012-4902

Abstract 1173: AKT-mTOR pathway mediates mutant p53 gain-of-function by inhibiting autophagy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Nearly half of human cancers harbor p53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. Persistent evidence supports that the mutant p53 proteins not only lose tumor suppressor activity, but also acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. Indeed, knock-in mouse models bearing the mutant p53R172H (equivalent to human R175H mutation) at physiological level in vivo display gain-of-function phenotypes, such as enhanced cell transformation potentials and tumor metastasis. However, the molecular mechanism of mutated p53R172H conferring gain-of-function remains poorly understood. In this study, utilizing mouse embryonic fibroblasts (MEFs) and lung adenocarcinoma cells (93-1) derived from p53R172H knock-in mice, we observed that AKT was preferentially activated in p53R172H mutant cells over p53-deficient cells. This activation of AKT allows p53R172H mutant cells to bypass contact inhibition, grow in an anchorage-independent manner and form tumors after subcutaneous injection into SCID mice. In contrast, expression of dominant negative AKT or shRNA-mediated AKT knockdown in p53R172H mutant cells inhibited colony formation in soft-agar and suppressed tumor growth in mouse xenograft tumor model. To directly dissect the essential role of AKT in p53R172H mutant-initiated tumorigenesis in vivo, we bred AKT1 knockout mice to p53R172H mutant knock-in mice. Loss of AKT1 significantly reduced the incidence of tumor development and extended the life span from around 3 months in AKT1-proficient and p53R172H mutant mice (AKT1+/+; p53R172H/R172H) to about 12 months in AKT1-deficient and p53R172H mutant mice (AKT1−/−; p53R172H/R172H). To understand how the activated AKT conferred mutant p53 gain-of-function, we demonstrated that p53R172H mutant activated AKT-mTOR signaling pathway that led to an inhibition of autophagy both in vitro and in vivo. Furthermore, induction of autophagy in p53R172H mutant lung cancer cells through suppressing AKT-mTOR pathway pharmacologically by the dual kinase inhibitor NVP-BEZ235 treatment or genetically by RNAi-mediated knockdown of mTOR dramatically inhibited p53R172H mutant-induced colony formation in soft agar and tumor growth in SCID mice. Our results support that AKT plays a crucial role in mediating mutant p53 gain-of-function phenotypes in vivo through the inhibition of autophagy by the activated AKT-mTOR signaling pathway. These findings provide the proof-of-principle that targeting the AKT-mTOR-autophagy pathway might have promising therapeutic benefits for cancer patients carrying such p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1173. doi:1538-7445.AM2012-1173

Abstract 3213: Selective killing of advanced prostate cancer cells by targeting the Warburg effect and autophagy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mutations of multiple tumor suppressor genes, such as PTEN and p53, have been proposed to play important roles in the development of prostate cancer. Loss of one allele of PTEN occurs in 70-80% of human primary prostate tumors and homozygous inactivation of PTEN is associated with advanced disease. Similarly, p53 is found completely lost or mutated almost exclusively in advanced human prostate cancer. Thus, selective killing of prostate cancer cells harboring mutations of PTEN and p53 may prove to be a promising strategy for the treatment of advanced prostate cancer. The Warburg effect of aerobic glycolysis has now been generally accepted as a key metabolic hallmark of cancer. In this study, we investigated the molecular target leading to the Warburg effect in the growth and aggressiveness of prostate cancer cells harboring inactivation of PTEN and p53 and delineated the underlying mechanism. We identified that expression of hexokinase II (HK2), an enzyme involved in the first step of glycolysis, is preferentially elevated in human prostate cancer cells bearing mutations of both PTEN and p53 (PC3). Functional studies demonstrated that HK2 expression is crucial for the Warburg effect in PC3 cells and knockdown of HK2 inhibits tumor growth in PC3 xenograft mouse model. These novel findings prompted us to test whether 2-de-oxyglucose (2-DG), an inhibitor of HK2, could potentially suppress prostate cancer growth by targeting the Warburg effect. We found that an induction of AMPK-dependent autophagy prevents cancer cells from apoptosis upon 2-DG treatment, thereby limiting therapeutic efficacy on prostate cancer in vivo. Consistent with cell survival function of autophagy, its inhibition by chloroquine (a small molecule inhibitor of autophagy) or individual knockdown of the essential genes involved in autophagy (Atg5, Atg7, Beclin1, and ULK1) induced massive cell death when combined with 2-DG. This cell death can be rescued by overexpression of anti-apoptotic protein Bcl-2 or downregulation of pro-apoptotic protein Bax in PC3. More importantly, we demonstrated that combination of chloroquine and 2-DG could specifically kill prostate cancer cells, leaving normal prostate epithelial cells untouched. This specificity is due to the preferential induction of HK2 through the activated Rictor-AKT-mTOR pathway in cancer cells. Finally, combination of chloroquine and 2-DG caused synthetic lethality in prostate cancer cells and effectively suppressed tumor growth in PC3 xenograft mouse model. Towards therapeutic translation, we have observed that expression of HK2 by staining primary human prostate tumor samples with HK2 antibodies correlated with the stages of prostate cancer. Given our findings, we therefore propose that targeting the Warburg effect and autophagy pathways may serve as an effective and selective treatment for patients with advanced prostate cancer, in particular those with PTEN and p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3213. doi:1538-7445.AM2012-3213