Katherine M. Aird

Synthetic lethality by targeting EZH2 methyltransferase activity in ARID1A -mutated cancers

ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.The gene encoding ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently have no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A-mutated ovarian cancer cells and that ARID1A mutational status correlated with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct target of ARID1A and EZH2 that is upregulated by EZH2 inhibition and contributed to the observed synthetic lethality by inhibiting PI3K–AKT signaling. Importantly, EZH2 inhibition caused regression of ARID1A-mutated ovarian tumors in vivo. To our knowledge, this is the first data set to demonstrate a synthetic lethality between ARID1A mutation and EZH2 inhibition. Our data indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for cancers involving ARID1A mutations.

Suppression of Nucleotide Metabolism Underlies the Establishment and Maintenance of Oncogene-Induced Senescence

Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here, we show that a decrease in deoxyribonucleotide triphosphate (dNTP) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of ribonucleotide reductase subunit M2 (RRM2), a rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell-cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Furthermore, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.

sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

Trastuzumab signaling in ErbB2-overexpressing inflammatory breast cancer correlates with X-linked inhibitor of apoptosis protein expression.

Inflammatory breast cancer (IBC) patients show poor survival and a significant incidence of epidermal growth factor receptor-2 (ErbB2) overexpression. A distinct mechanism involving increased expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin, key members of the inhibitor of apoptosis protein (IAP) family, was observed post-trastuzumab (an ErbB2 monoclonal antibody) treatment in an ErbB2-overexpressing, estrogen receptor negative, IBC cellular model, SUM190PT, isolated from a primary IBC tumor. In contrast, a decrease in the IAP expression was observed in the non-IBC, ErbB2-overexpressing SKBR3 cells in which trastuzumab treatment also decreased p-AKT and cell viability. Further, in SUM190PT cells, therapeutic sensitivity to GW583340 (a dual epidermal growth factor receptor/ErbB2 kinase inhibitor) corresponded with XIAP down-regulation and abrogation of XIAP inhibition on active caspase-9 release. Specific small interfering RNA–mediated XIAP inhibition in combination with trastuzumab caused decrease in inactive procaspase-9 and inhibition of p-AKT corresponding with 45% to 50% decrease in cell viability in the SUM190PT cells, which have high steady-state p-AKT levels. Further, embelin, a small-molecule inhibitor that abrogates binding of XIAP to procaspase-9, caused significant decrease in SUM190PT viability. However, embelin in combination with trastuzumab failed to affect SUM190PT viability because it has no direct effect on XIAP, which is induced by trastuzumab treatment. These data have identified a novel functional link between ErbB2 signaling and antiapoptotic pathway mediated by XIAP. Blockade of the IAP antiapoptotic pathway alone or in combination would be an attractive strategy in IBC therapy. [Mol Cancer Ther 2008;7(1):38–47]

Susceptibility to Tuberculosis: Clues from Studies with Inbred and Outbred New Zealand White Rabbits

ABSTRACT The rabbit model of tuberculosis (TB) is important because rabbits develop a disease that is similar to TB in humans, namely, granulomas with caseous necrosis, liquefaction, and cavities. We describe here a comparison of inbred and outbred New Zealand White rabbits infected by aerosol with either Mycobacterium tuberculosis Erdman or H37Rv strain. Five weeks after infection with either bacillary strain, the inbred rabbits had significantly larger pulmonary tubercles than did outbred rabbits (2.7 versus 1.4 mm in diameter; P < 0.01). After infection with H37Rv, the inbred rabbits had significantly more pulmonary tubercles than did the outbred rabbits (98 ± 12 versus 33 ± 13; P < 0.01), with more mycobacterial CFU per tubercle (809 ± 210 versus 215 ± 115; P = 0.027) (means ± standard errors of the means). Compared with histologic examination of lung granulomas from outbred rabbits, histologic examination of those from inbred rabbits showed more caseous necrosis, more visible bacilli, and fewer mature epithelioid cells. The delayed-type hypersensitivity (DTH) responses to intradermal tuberculin were significantly lower, and peritoneal macrophages from uninfected inbred rabbits produced significantly less tumor necrosis factor alpha after lipopolysaccharide (LPS) stimulation in vitro than those from the outbred rabbits (2,413 ± 1,154 versus 8,879 ± 966 pg/ml). We conclude that these inbred rabbits were more susceptible to TB than their outbred counterparts and had an impaired ability to contain disease, resulting in more grossly visible tubercles that were larger than those observed in outbred rabbits. Preliminary evidence is presented for a cell-mediated immune defect with lower DTH responses and macrophages that have a decreased ability to respond to in vitro stimulation with LPS or M. tuberculosis infection.

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

Significance Despite the promise of personalized cancer medicine, most molecular therapies produce only modest and short-lived patient gains. In addition to drug resistance, it is also possible that tumors adaptively reprogram their signaling pathways to evade therapy-induced “stress” and, in the process, acquire more aggressive disease traits. We show here that small-molecule inhibitors of PI3K, a cancer node and important therapeutic target, induce transcriptional and signaling reprogramming in tumors. This involves the trafficking of energetically active mitochondria to subcellular sites of cell motility, where they provide a potent, “regional” energy source to support tumor cell invasion. Although this response may paradoxically increase the risk of metastasis during PI3K therapy, targeting mitochondrial reprogramming is feasible, and could provide a novel therapeutic strategy. Molecular therapies are hallmarks of “personalized” medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, “spatiotemporal” mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

X-Linked Inhibitor of Apoptosis Protein Inhibits Apoptosis in Inflammatory Breast Cancer Cells with Acquired Resistance to an ErbB1/2 Tyrosine Kinase Inhibitor

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small-molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analogue, ErbB1/2 inhibitor)–resistant IBC cells, we identified the overexpression of an antiapoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), in acquired resistance to GW583340 in both ErbB2-overexpressing SUM190 and ErbB1-activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting that the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small-molecule inhibitor (embelin), which abrogates the XIAP/procaspase-9 interaction, resulted in decreased viability, showing that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents. Mol Cancer Ther; 9(5); 1432–42. ©2010 AACR.

XIAP Inhibition and Generation of Reactive Oxygen Species Enhances TRAIL Sensitivity in Inflammatory Breast Cancer Cells

We recently identified superoxide dismutase (SOD) overexpression and decreased induction of reactive oxygen species (ROS)-mediated apoptosis in models of inflammatory breast cancer (IBC) cells with acquired therapeutic resistance. This population of cells has high expression of X-linked inhibitor of apoptosis protein (XIAP), which inhibits both extrinsic and intrinsic apoptosis pathways. We therefore wanted to evaluate the effect of classical apoptosis-inducing agent TRAIL, a proapoptotic receptor agonist that selectively triggers death receptor (DR)-mediated apoptosis in cancer cells, in the IBC acquired resistance model. XIAP levels and subsequent inhibition of caspase activity inversely correlated with TRAIL sensitivity in our models of IBC. These include SUM149, a basal-type cell line isolated from primary IBC tumors and isogenic SUM149-derived lines rSUM149 and SUM149 wtXIAP, models of acquired therapeutic resistance with endogenous and exogenous XIAP overexpression, respectively. Inhibition of XIAP function using embelin, a plant-derived cell permeable small molecule, in combination with TRAIL caused a synergistic decrease in cell viability. Embelin treatment resulted in activation of extracellular signal–regulated kinase (ERK)1/2 and ROS accumulation, which correlated with downregulation of antioxidant protein SOD1 and consumption of redox modulator reduced glutathione in the XIAP-overexpressing cells. Simultaneous treatment with an SOD mimic, which protects against ROS accumulation, reversed the decrease in cell viability caused by embelin + TRAIL treatment. Embelin primes IBC cells for TRAIL-mediated apoptosis by its direct action on the anti-caspase activity of XIAP and by shifting the cellular redox balance toward oxidative stress–mediated apoptosis. Thus, ROS modulators represent a novel approach to enhance efficacy of TRAIL-based treatment protocols in IBC. Mol Cancer Ther; 11(7); 1518–27. ©2012 AACR.

Cytotoxic effects of Mn(III) N-alkylpyridylporphyrins in the presence of cellular reductant, ascorbate

Abstract Due to the ability to easily accept and donate electrons Mn(III)N-alkylpyridylporphyrins (MnPs) can dismute O2 ·−, reduce peroxynitrite, but also generate reactive species and behave as pro-oxidants if conditions favour such action. Herein two ortho isomers, MnTE-2-PyP5+, MnTnHex-2-PyP5+, and a meta isomer MnTnHex-3-PyP5+, which differ greatly with regard to their metal-centered reduction potential, E1/2 (MnIIIP/MnIIP) and lipophilicity, were explored. Employing MnIIIP/MnIIP redox system for coupling with ascorbate, these MnPs catalyze ascorbate oxidation and thus peroxide production. Consequently, cancer oxidative burden may be enhanced, which in turn would suppress its growth. Cytotoxic effects on Caco-2, Hela, 4T1, HCT116 and SUM149 were studied. When combined with ascorbate, MnPs killed cancer cells via peroxide produced outside of the cell. MnTE-2-PyP5+ was the most efficacious catalyst for peroxide production, while MnTnHex-3-PyP5+ is most prone to oxidative degradation with H2 , and thus the least efficacious. A 4T1 breast cancer mouse study of limited scope and success was conducted. The tumour oxidative stress was enhanced and its microvessel density reduced when mice were treated either with ascorbate or MnP/ascorbate; the trend towards tumour growth suppression was detected.

Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration.

Background:Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients.Methods:The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays.Results:Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.Conclusion:Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.