Ciara H. O'Flanagan

The Parkinson’s gene PINK1 regulates cell cycle progression and promotes cancer-associated phenotypes

PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson’s disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1−/− mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1−/− mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1−/− cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.

PINK1 signalling in cancer biology

PTEN-induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up-regulated by overexpression of the major tumor suppressor, PTEN. Loss-of-function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsons disease. Substantial work during the past decade has revealed that PINK1 regulates several primary cellular processes of significance in cancer cell biology, including cell survival, stress resistance, mitochondrial homeostasis and the cell cycle. Mechanistically, PINK1 has been shown to interact on a number of levels with the pivotal oncogenic PI3-kinase/Akt/mTOR signalling axis and to control critical mitochondrial and metabolic functions that regulate cancer survival, growth, stress resistance and the cell cycle. A cytoprotective and chemoresistant function for PINK1 has been highlighted by some studies, supporting PINK1 as a target in cancer therapeutics. This article reviews the function of PINK1 in cancer cell biology, with an emphasis on the mechanisms by which PINK1 interacts with PI3-kinase/Akt signalling, mitochondrial homeostasis, and the potential context-dependent pro- and anti-tumorigenic functions of PINK1.

Abstract 1794: The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion

Insulin-like Growth Factor-1 signaling has a well-described function in facilitating tumourigenesis and promoting tumour growth. Attempts to suppress IGF signals at the level of the IGF-1 Receptor have been disappointing, with kinase inhibitors and blocking antibodies generally showing poor efficacy in clinical trials. To address the mechanisms for this lack of efficacy we sought to identify proteins that modulate the cytotoxic response to IGF-1R tyrosine kinase inhibition using a functional siRNA screen. We identified the non-receptor tyrosine FES-related (FER) kinase as a mediator of sensitivity to the IGF-1R tyrosine kinase inhibitor in MCF-7 cells. We found that FER and the IGF-1R co-locate and can be co-immunoprecipitated from different cell types. Ectopic expression of FER strongly enhanced IGF-1R expression and phosphorylation on the atuophosphorylation sites at tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either the AKT or MAPK signaling pathways in breast cancer cell lines. The IGF-1R, β1 Integrin, FER and its substrate cortactin were all observed to be co-located in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known ALK inhibitor that shows high specificity for FER kinase. Overall, we conclude that FER-enhances IGF-1R expression, phosphorylation and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression and resistance to IGF-1R inhibition. Citation Format: Joanna Stanicka, Leonie Rieger, Orla T. Cox, Sandra O9Shea, Michael Coleman, Ciara O9Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O9Connor. The FES-related tyrosine kinase associates with and activates the insulin-like growth factor 1 receptor at sites of cell adhesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1794.

Abstract 247: Nutrient stress via folic acid modulation causes systemic and cancer-specific metabolic reprogramming and differential effects on primary and metastatic mammary tumor growth in lean and obese mice

Many foods are currently fortified with folic acid (FA), a synthetic folate (Vitamin B9). Folate deficiency causes many human health defects, most notably anemia and neural tube defects. The effects of excess supplementation on human health have to date been understudied. The rise in obesity in the last three decades further complicates this issue, with the combined effects of excess or insufficient folic acid intake and an obese phenotype being unknown. Obesity is associated with a number of cancers, including triple negative breast cancer (TNBC). TNBC comprises 16% of all breast cancers, is highly aggressive and more likely to recur and metastasize than other breast cancers. Unlike other subtypes, TNBC does not respond to hormone-targeted therapies and treatment options are limited to cytotoxic chemotherapy. Here, we examined the effects of FA supplementation and deficiency on tumor growth, metastasis and metabolism in obesity-responsive models of primary (M-Wnt) and metastatic (metM-Wnt; MDA-MB-231) TNBC. FA supplementation and deficiency significantly enhanced primary tumor growth and invasiveness in lean mice, while no difference in tumor size was detected in obese groups. FA supplementation reduced while deficiency increased survival and reduced lung tumor metastasis incidence in lean, but not obese mice. Liver and tumor metabolomic profiling revealed that modulation of dietary FA caused systemic and tumor-specific metabolic reprogramming, altering pathways involved in fatty acid, purine, amino acid, glutathione and energy metabolism. Short term in vitro FA withdrawal resulted in reduced proliferation, migration and invasion and energy production in all cell lines, as well as significant changes in gene expression profile, particularly of many metabolic pathways. In contrast, chronic in vitro FA depletion resulted in heightened oxidative stress, autophagy and apoptosis in metastatic TNBC cells, with nonmetastatic TNBC cells being able to adapt to and withstand the nutrient stress via the pentose phosphate pathway and glutathione redox signaling. Taken together, these results suggest that modulation of dietary folic acid in lean (but not obese) individuals causes systemic and tumor-specific metabolic reprogramming, which may confer a growth advantage in nonmetastatic cells and from which metastatic TNBC cells cannot recover. Moreover, obesity and FA excess cause similar metabolic and procancer effects and in combination, are not synergistic. Citation Format: Ciara H. O9Flanagan, Xuewen Chen, Zahra Ashkavand, Sergey A. Krupenko, Stephen D. Hursting. Nutrient stress via folic acid modulation causes systemic and cancer-specific metabolic reprogramming and differential effects on primary and metastatic mammary tumor growth in lean and obese mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 247. doi:10.1158/1538-7445.AM2017-247

Abstract 446: Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells

Folate coenzymes play an important role in biosynthesis and methylation reactions. Aberrant folate metabolism has been implicated in the development of several cancer types, though mechanisms underlying folate metabolism and cancer development remain unclear. We previously showed that a folate-restricted diet can exert differential effects on metastatic versus nonmetastatic murine claudin-low breast cancer cells in vivo. Specifically, folate restriction increased growth and invasion of orthotopically transplanted M-Wnt (nonmetastatic) tumor cells, but decreased growth and lung metastases of transplanted metM-Wnt cells, a metastatic subclone of M-Wnt cells. The current study set out to explore the underlying mechanism. To examine the effect of long-term folate depletion (LFD) on M-Wnt and metM-Wnt cell metabolism, oxidative stress and autophagic flux in vitro, the two cell lines were grown in standard and folate-depleted media for 14 days. metM-Wnt cells showed higher oxidative stress, as measured by ROS staining and Nrf2 expression, and phosphorylation of the key nutrient sensor, AMPK, compared to M-Wnt cells when grown in standard growth medium. LFD M-Wnt cells showed an increased dependence on glycolysis compared to those cultured in standard medium. Both M-Wnt and metM-Wnt cells displayed a high autophagy level in LFD, measured by LC3B cleavage, and AMPK phosphorylation. However, LFD metM-Wnt cells showed low viability, increased apoptosis and loss of redox defense, as measured by cleaved-caspase 3 and Nrf2 expressions. These results suggest that non-metastatic M-Wnt cells undergo metabolic reprogramming, including a shift from oxidative phosphorylation to glycolysis that may fuel cell growth and proliferation. Further, an elevated autophagic flux may mitigate nutrient stress induced by folate depletion, which allows them to withstand LFD and which may contribute to a more invasive primary tumor in response to folate restriction. In contrast, metM-Wnt cells are unable to undergo this metabolic adaptation, and display increased oxidative stress and cell death in response to LFD, preventing the development of metastatic lesions in vitro. This study highlights different responses of primary and metastatic breast cancer cells to folate depletion. The results provide additional rationale for targeting folate metabolism as a potential strategy for treating metastatic breast cancer. Citation Format: Xuewen Chen, Ciara H. O9Flanagan, Stephen D. Hursting. Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2017-446

Abstract 4082: PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition

PDLIM2 is a PDZ-LIM domain protein that regulates the stability and activity of transcription factor families (including NFκB, STATs and beta catenin). PDLIM2 expression is repressed in certain cancers but it is also highly expressed in Triple Negative Basal Breast cancers that are characterized by poor survival. Suppression of PDLIM2 reverses the EMT phenotype, inhibits polarized cell migration, and disrupts formation of polarized epithelial acini in 3D cell cultures. PDLIM2 shuttles from the cytoskeleton to the nucleus, but what mediates this nuclear translocation or activity in transcription factor regulation is unknown. The aim of this study was to identify the mechanisms governing PDLIM2 subcellular localization and nuclear translocation. We found that IGF-1or TGF-β promotes PDLIM2 accumulation in the nucleus. Similarly, WNT3a stimulation enhances PDLIM2 accumulation in the nucleus while inhibition of WNT activity results in PDLIM2 stabilization in the cytoplasm. Cytoplasmic to nuclear translocation is associated with reduced phosphorylation on several serine residues in PDLIM2. The de-phosphorylation and subsequent nuclear translocation of PDLIM2 can be prevented by inhibiting the protein phosphatase PP1. In contrast, PDLIM2 phosphorylation can be enhanced by activation of protein kinase C, which is dependent on the presence of the focal adhesion scaffolding protein RACK1 in a complex with PDLIM2. Overall, the data indicate that PDLIM2 cytoplasmic to nuclear translocation in response to IGF-1, WNT or TGF beta signalling is mediated by serine phosphorylation and de-phosphorylation by cytoskeleton-associated kinases and phosphatases. Thus PDLIM2 acts as a “cytoskeleton to nucleus” courier protein for these signalling pathways to promote cancer cell migration and EMT. Citation Format: Milan Bustamante Garrido, Orla T. Cox, Ciara O9Flanagan, Deirdre A. Buckley, Patrick A. Kiely, Rosemary O9Connor. PDLIM2 : A cytoskeleton to nuclear courier protein for the IGF-1, Wnt and TGF beta signalling pathways in Epithelial to Mesenchymal Transition. [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 4082. doi:10.1158/1538-7445.AM2015-4082

Abstract 1117: Investigating the role of PTEN-induced kinase 1 in cancer development and pathogenesis

PTEN-induced kinase 1 (PINK1) was first identified in cancer cells as a gene up-regulated by overexpression of the central tumour suppressor, PTEN. Loss-of-function mutations in PINK1 were subsequently discovered to cause autosomal recessive Parkinson9s disease (ARPD). While much research has focused on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, some studies indicate a potential role for this serine/threonine kinase in cancer cell biology. PINK1 is known to be a pro-survival kinase, protecting cells from several stressors, and is a key controller of mitochondrial integrity, morphology and function. This study aimed to understand the function of PINK1 in the development of cancer. Using SV40 immortalised PINK1 -/- mouse embryonic fibroblasts (MEFs), we show that loss of PINK1 results in reduced cell proliferation and foci formation, as well as decreased invasiveness. Overexpression of human PINK1 in these cells rescues these phenotypes and increases migratory capacity, indicating a tumour promoting role for PINK1. Furthermore, PINK1 loss increases autophagy and levels of reactive oxygen species, as well as enhancing mitochondrial fission. Notably, we identify PINK1 as a novel controller of cell cycle progression events which are critical to tumourigenesis. These results indicate an important function for PINK1 in cancer cell development. 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 1117. doi:1538-7445.AM2012-1117