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Supplementary Table I (RPPA) and Table II (Gene expression data).

Supplementary Methods

Related to Figure 6: Western blots and immunofluorescence images of PDLIM2 and b-catenin phospho-Ser675 expression and localization in BT549 cells overexpressing HA-PDLIM2 are included. Graph depicting measurements of cell migration in HA-PDLIM2-expressing BT549 is shown. The effects of suppression of PDLIM2 on b-catenin and b1-integrin levels in HCC1806 and MDA-MB-231-LUC2 are shown. Further analyses of effects of loss of adhesion and also, serum starvation, on PDLIM2 and active b-catenin subcellular localization in TN Breast cell lines are also shown.

The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and β-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFβ. Cytoplasmic PDLIM2 was associated with active β-catenin and ectopic expression of PDLIM2 was sufficient to increase β-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated β-catenin activity and which may be amenable to therapies that target these pathways.Significance:This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the β-catenin and adhesion signaling pathways.

Related to Figures 5 and 6: Contains images of PDLIM2 immunocytochemistry staining of PDLIM2-negative TNBC cell lines not included in Figure 5 and additional analysis of TN Breast cells under adhesion versus suspension conditions. Protein expression and quantification of β-catenin phosphorylated on Ser675 are also shown. Immunofluorescence images of HA-PDLIM2 overexpressed in BT549 cells are included.

Related to Figure 2: Shows data for distribution of IHC scores of PDLIM2 staining in TNBC tissues and examples of PDLIM2 IHC images of normal tissue. Includes Kaplan Meier curves and clinicopathological analysis of RATHER cohorts in relation to PDLIM2 expression.

Related to Figures 2 and 3: Shows counts of distribution of IHC scores of PDLIM2 staining in RATHER cohort based on TNBC subtype. Includes analysis of TNBC subtypes of tissues in the RATHER cohorts in relation to PDLIM2 expression, a Kaplan Meier plot of breast cancer specific survival in relation to TNBC subtype, and correlation plot of PDLIM2 mRNA and protein expression in the RATHER cohorts. Examples of nuclear PDLIM2 expression in stromal/infiltrating cells are also shown.

Related to Figure 1: Includes additional images of immunohistochemical staining and scores of PDLIM2 expression in tumor and stroma of Breast Cancer TMA samples.

Related to Figure 4: Includes TNBC subtype classification of the cell lines used in this study and additional analyses of expression of PDLIM2 and epithelial/mesenchymal markers. A graph illustrates PDLIM2 RNASeq and protein expression in the cell lines. Expression of b1 integrin, Phospho-EGFR, Phospho-IGF-1R and Phospho c-Met in TN Breast cell lines are also shown.

We have previously shown that withdrawal of folic acid led to metabolic reprogramming and a less aggressive phenotype in a mouse cell model of triple-negative breast cancer (TNBC). Herein, we evaluate the effects of folic acid withdrawal on transcriptomic profiles in these cells. Murine cell lines were originally derived from a pool of spontaneous mammary tumors grown in MMTV-Wnt1 transgenic mice. Based on their differential molecular characteristics and metastatic potential, these cell lines were previously characterized as non-metastatic epithelial (E-Wnt), non-metastatic mesenchymal (M-Wnt) and metastatic mesenchymal (metM-Wnt(liver)) cells. Using custom two-color 180K Agilent microarrays, we have determined gene expression profiles for three biological replicates of each subtype kept on standard medium (2.2 μM folic acid) or folic acid-free medium for 72 hours. The analyses revealed that more genes were differentially expressed upon folic acid withdrawal in M-Wnt cells (1884 genes; Benjamini-Hochberg-adjusted P-value

Cancer genomes exhibit extensive chromosomal copy number changes and structural variation, yet how allele specific alterations drive cancer genome evolution remains unclear. Here, through application of a new computational approach we report allele specific copy number alterations in 11,097 single cell whole genomes from genetically engineered mammary epithelial cells and 21,852 cells from high grade serous ovarian and triple negative breast cancers. Resolving single cell copy number profiles to individual alleles uncovered genomic background distributions of gains, losses and loss of heterozygosity, yielding evidence of positive selection of specific chromosomal alterations. In addition specific genomic loci in maternal and paternal alleles were commonly found to be altered in parallel with convergent phenotypic transcriptional effects. Finally we show that haplotype specific alterations trace the cyclical etiology of high level amplifications and reveal clonal haplotype decomposition of complex structures. Together, our results illuminate how allele and haplotype specific alterations, here determined across thousands of single cell cancer genomes, impact the etiology and evolution of structural variations in human tumours.

Structural genome alterations are determinants of cancer ontogeny and therapeutic response. While bulk genome sequencing has enabled delineation of structural variation (SV) mutational processes which generate patterns of DNA damage, we have little understanding of how these processes lead to cell-to-cell variations which underlie selection and rates of accrual of different genomic lesions. We analysed 309 high grade serous ovarian and triple negative breast cancer genomes to determine their mutational processes, selecting 22 from which we sequenced >22,000 single cell whole genomes across a spectrum of mutational processes. We show that distinct patterns of cell-to-cell variation in aneuploidy, copy number alteration (CNA) and segment length occur in homologous recombination deficiency (HRD) and fold-back inversion (FBI) phenotypes. Widespread aneuploidy through induction of HRD throughBRCA1andBRCA2inactivation was mirrored by continuous whole genome duplication in HRD tumours, contrasted with early ploidy fixation in FBI. FBI tumours exhibited copy number distributions skewed towards gains, widespread clone-specific variation in amplitude of high-level amplifications, often impacting oncogenes, and break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). SSVs were consistent with a CNA-based molecular clock reflecting a continual and distributed process across clones within tumours. These observations reveal previously obscured genome plasticity and evolutionary properties with implications for cancer evolution, therapeutic targeting and response.

How cell-to-cell copy number alterations that underpin genomic instability

Measuring gene expression of tumor clones at single-cell resolution links functional consequences to somatic alterations. Without scalable methods to simultaneously assay DNA and RNA from the same single cell, parallel single-cell DNA and RNA measurements from independent cell populations must be mapped for genome-transcriptome association. We present clonealign, which assigns gene expression states to cancer clones using single-cell RNA and DNA sequencing independently sampled from a heterogeneous population. We apply clonealign to triple-negative breast cancer patient-derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either sequencing method alone. Electronic supplementary material The online version of this article (10.1186/s13059-019-1645-z) contains supplementary material, which is available to authorized users.

Copy number alterations and structural variants are associated with disease progression, therapeutic response, and metastasis in human cancers, yet the extent and mechanisms driving continued genomic instability remain poorly understood. We generated more than 20,000 single-cell whole genomes from 25 high-grade serous ovarian and triple-negative breast cancers across a range of mutational subtypes. Through the development of new computational methods, we resolved alterations into cancer haplotypes, providing fine-grained resolution of genomic events in single-cells and exposing extensive cell-to-cell genomic variation. We identified 3 key processes that contribute to this diversity. Firstly, we observed that parallel copy number events - whereby different cancer cells harbor chromosomal aberrations with identical total copy number but different combinations of maternal and paternal alleles - were present in > 80% of the tumors. This is consistent with extensive convergent evolution in these tumors. Secondly, we identified considerable cell-to-cell variability in the amplitude of high-level amplifications, often impacting oncogenes. Single-cell transcriptomes and immunohistochemistry from the same samples showed that this genomic diversity led to phenotypic differences between cells. Finally, we observed copy number break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). Both SSV’s and variable high-level amplifications were enriched in foldback inversion (FBI) bearing tumors relative to homologous recombination deficient tumors, highlighting that FBI tumors have greater levels of structural instability. Our study sheds new light on the evolution of genomically unstable tumors with implications for cancer evolution, therapeutic targeting, and patient stratification. Citation Format: Marc J. Williams, Tyler Funnell, Ciara H. O'Flanagan, Andrew McPherson, Samuel Aparicio, Sohrab P. Shah. Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1597.

Background: Triple-negative breast cancers (TNBCs), accounting for approximately 15% of breast cancers, lack targeted therapy. A hallmark of cancer is metabolic reprogramming, with one-carbon metabolism essential to many processes altered in tumor cells, including nucleotide biosynthesis and antioxidant defenses. We reported that folate deficiency via folic acid (FA) withdrawal in several TNBC cell lines results in heterogenous effects on cell growth, metabolic reprogramming, and mitochondrial impairment. To elucidate underlying drivers of TNBC sensitivity to folate stress, we characterized in vivo and in vitro responses to FA restriction in two TNBC models differing in metastatic potential and innate mitochondrial dysfunction. Methods: Metastatic MDA-MB-231 cells (high mitochondrial dysfunction) and nonmetastatic M-Wnt cells (low mitochondrial dysfunction) were orthotopically injected into mice fed diets with either 2 ppm FA (control), 0 ppm FA, or 12 ppm FA (supplementation, in MDA-MB-231 only). Tumor growth, metabolomics, and metabolic gene expression were assessed. MDA-MB-231 and M-Wnt cells were also grown in media with 0 or 2.2 µM FA, metabolic alterations were assessed by extracellular flux analysis, flow cytometry, and qPCR. Results: Relative to control, dietary FA restriction decreased MDA-MB-231 tumor weight and volume, while FA supplementation minimally increased MDA-MB-231 tumor weight. Metabolic studies in vivo and in vitro using MDA-MB-231 cells showed FA restriction remodeled one-carbon metabolism, nucleotide biosynthesis, and glucose metabolism. In contrast to findings in the MDA-MB-231 model, FA restriction in the M-Wnt model, relative to control, led to accelerated tumor growth, minimal metabolic changes, and modest mitochondrial dysfunction. Increased mitochondrial dysfunction in M-Wnt cells, induced via chloramphenicol, significantly enhanced responsiveness to the cytotoxic effects of FA restriction. Conclusions: Given the lack of targeted treatment options for TNBC, uncovering metabolic vulnerabilities that can be exploited as therapeutic targets is an important goal. Our findings suggest that a major driver of TNBC sensitivity to folate restriction is a high innate level of mitochondrial dysfunction, which can increase dependence on one-carbon metabolism. Thus, folate deprivation or antifolate therapy for TNBCs with metabolic inflexibility due to their elevated levels of mitochondrial dysfunction may represent a novel precision-medicine strategy.

Tumour fitness landscapes underpin selection in cancer, impacting etiology, evolution and response to treatment. Progress in defining fitness landscapes has been impeded by a lack of timeseries perturbation experiments over realistic intervals at single cell resolution. We studied the nature of clonal dynamics induced by genetic and pharmacologic perturbation with a quantitative fitness model developed to ascribe quantitative selective coefficients to individual cancer clones, enable prediction of clone-specific growth potential, and forecast competitive clonal dynamics over time. We applied the model to serial single cell genome (>60,000 cells) and transcriptome (>58,000 cells) experiments ranging from 10 months to 2.5 years in duration. We found that genetic perturbation ofTP53in epithelial cell lines induces multiple forms of copy number alteration that confer increased fitness to clonal populations with measurable consequences on gene expression. In patient derived xenografts, predicted selective coefficients accurately forecasted clonal competition dynamics, that were validated with timeseries sampling of experimentally engineered mixtures of low and high fitness clones. In cisplatin-treated patient derived xenografts, the fitness landscape was inverted in a time-dependent manner, whereby a drug resistant clone emerged from a phylogenetic lineage of low fitness clones, and high fitness clones were eradicated. Moreover, clonal selection mediated reversible drug response early in the selection process, whereas late dynamics in genomically fixed clones were associated with transcriptional plasticity on a fixed clonal genotype. Together, our findings outline causal mechanisms with implication for interpreting how mutations and multi-faceted drug resistance mechanisms shape the etiology and cellular fitness of human cancers.

Calorie restriction (CR) extends lifespan and has been shown to reduce age-related diseases including cancer, diabetes, and cardiovascular and neurodegenerative diseases in experimental models. Recent translational studies have tested the potential of CR or CR mimetics as adjuvant therapies to enhance the efficacy of chemotherapy, radiation therapy, and novel immunotherapies. Chronic CR is challenging to employ in cancer patients, and therefore intermittent fasting, CR mimetic drugs, or alternative diets (such as a ketogenic diet), may be more suitable. Intermittent fasting has been shown to enhance treatment with both chemotherapy and radiation therapy. CR and fasting elicit different responses in normal and cancer cells, and reduce certain side effects of cytotoxic therapy. Findings from preclinical studies of CR mimetic drugs and other dietary interventions, such as the ketogenic diet, are promising for improving the efficacy of anticancer therapies and reducing the side effects of cytotoxic treatments. Current and future clinical studies will inform on which cancers, and at which stage of the cancer process, CR, fasting, or CR mimetic regimens will prove most effective.

Folate coenzymes are involved in biochemical reactions of one-carbon transfer, and deficiency of this vitamin impairs cellular proliferation, migration, and survival in many cell types. Here, the effect of folate restriction on mammary cancer was evaluated using three distinct breast cancer subtypes differing in their aggressiveness and metastatic potential: noninvasive basal-like (E-Wnt), invasive but minimally metastatic claudin-low (M-Wnt), and highly metastatic claudin-low (metM-Wntliver) cell lines, each derived from the same pool of MMTV-Wnt-1 transgenic mouse mammary tumors. NMR-based metabolomics was used to quantitate 41 major metabolites in cells grown in folate-free medium versus standard medium. Each cell line demonstrated metabolic reprogramming when grown in folate-free medium. In E-Wnt, M-Wnt, and metM-Wntliver cells, 12, 29, and 25 metabolites, respectively, were significantly different (P < 0.05 and at least 1.5-fold change). The levels of eight metabolites (aspartate, ATP, creatine, creatine phosphate, formate, serine, taurine and β-alanine) were changed in each folate-restricted cell line. Increased glucose, decreased lactate, and inhibition of glycolysis, cellular proliferation, migration, and invasion occurred in M-Wnt and metM-Wntliver cells (but not E-Wnt cells) grown in folate-free versus standard medium. These effects were accompanied by altered levels of several folate-metabolizing enzymes, indicating that the observed metabolic reprogramming may result from both decreased folate availability and altered folate metabolism. These findings reveal that folate restriction results in metabolic and bioenergetic changes and a less aggressive cancer cell phenotype. Implications: Metabolic reprogramming driven by folate restriction represents a therapeutic target for reducing the burden of breast cancer. Mol Cancer Res; 15(2); 189–200. ©2016 AACR.

BackgroundSingle-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.ResultsWe observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically “dying” cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37°C or 6°C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, includingFOSandJUN, induced by collagenase (37°C), which are minimized by dissociation with a cold active protease (6°C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.ConclusionsThe method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and β-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFβ. Cytoplasmic PDLIM2 was associated with active β-catenin and ectopic expression of PDLIM2 was sufficient to increase β-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated β-catenin activity and which may be amenable to therapies that target these pathways. Significance: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the β-catenin and adhesion signaling pathways.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. Implications: These findings underlie the importance of a novel morphologic–metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

Single-cell RNA sequencing (scRNA-seq) has transformed biomedical research, enabling decomposition of complex tissues into disaggregated, functionally distinct cell types. For many applications, investigators wish to identify cell types with known marker genes. Typically, such cell type assignments are performed through unsupervised clustering followed by manual annotation based on these marker genes, or via "mapping" procedures to existing data. However, the manual interpretation required in the former case scales poorly to large datasets, which are also often prone to batch effects, while existing data for purified cell types must be available for the latter. Furthermore, unsupervised clustering can be error-prone, leading to under- and over- clustering of the cell types of interest. To overcome these issues we present CellAssign, a probabilistic model that leverages prior knowledge of cell type marker genes to annotate scRNA-seq data into pre-defined and de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while simultaneously controlling for batch and patient effects. We demonstrate the analytical advantages of CellAssign through extensive simulations and exemplify real-world utility to profile the spatial dynamics of high-grade serous ovarian cancer and the temporal dynamics of follicular lymphoma. Our analysis reveals subclonal malignant phenotypes and points towards an evolutionary interplay between immune and cancer cell populations with cancer cells escaping immune recognition.

Single-cell RNA sequencing (scRNA-seq) has enabled decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types, performed through unsupervised clustering followed by manual annotation, or via “mapping” procedures to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data, and both are prone to batch effects. To overcome these issues we present CellAssign (www.github.com/irrationone/cellassign), a probabilistic model that leverages prior knowledge of cell type marker genes to annotate scRNA-seq data into pre-defined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high grade serous ovarian cancer and follicular lymphoma.

Introduction Breast cancer is the most common cancer in female and triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer which shows high rate of recurrence and metastasis. Malignant cells that comprise primary tumor are heterogeneous and during disease progression selection of tumor cells occur as a mean to adapt and survive. Tumor heterogeneity can be studied by grouping cells as clones which refer to a group of cells related to each other by descent from a unitary origin. Understanding the mechanism of clonal dynamics and selection during cancer evolution is important to develop new therapeutic strategies for cancer metastasis. Moreover, it is imperative to be able to detect rare subclones that are responsible for metastasis. Thus, single-cell analysis is critical to identify cellular heterogeneity of cancer and molecular basis of metastatic phenotype in depth. Measuring genome and transcriptome in single-cell level will enable us to discover clonal dynamics during cancer metastasis and infer molecular determinants of metastasis fitness. Here, we propose to investigate the clonal dynamics, genomic, and transcriptomic profiles of human breast tumor metastasis using TNBC patient-derived xenograft (PDX) model to understand the mechanism of breast cancer progression and metastasis. Methods Tumor cells from previously established PDXs were transplanted into mammary fat pad of mice. Tumors were removed when it reached maximum allowed endpoint size (1,000mm3) and mice were monitored and allowed to grow metastasis. PDXs that developed primary tumor and metastasis were selected for subsequent single cell analysis. Single-cell whole-genome sequencing (scWGS) was performed using direct library preparation (DLP+) method which is preamplification-free single-cell genome sequencing approach. scWGS data was used to call cell-specific copy number events, which allows us to cluster cells and identify clones. Hierarchical clustering was used to identify clonal structure of each sample. Phylogenetic tree was computed using copy number data of samples from each PDX. Results Nine different TNBC PDX lines were tested and 6 PDX lines developed metastasis. We focused on 2 PDX lines of which metastatic tissues were available for single cell sequencing. SA919 developed metastatic mass near cervical or lumbar spine area. SA535 developed metastasis to lung, axillary or inguinal area and tumor recurrence at mammary fat pad site. scWGS for SA919 and SA535 was carried out and we generated libraries from single cells of primary tumor and its metastases. Hierarchical clustering using copy number data revealed clonal structure of primary tumor and metastases. SA919 showed oligoclonal primary tumor and metastasis whereas SA535 showed polyclonal metastasis from polyclonal primary tumor. scWGS data of primary tumor and its metastases in each PDX were merged to compute phylogenetic trees. Phylogenetic analysis revealed that not all clones of primary tumor contributed to metastasis in both PDXs. Different metastatic lesions of SA535 harbored different clones from primary tumor. For example, clones consisting left axillary metastasis and right axillary metastasis in SA535 were different from each other, however they both originated from primary tumor clones. Metastatic lesions from SA919 and SA535 also had newly appearing clones that were not present in primary tumor inferring clonal evolution during metastasis. Conclusion We were able to capture different patterns of metastasis in several PDXs. Phylogenetic analysis using scWGS data revealed clonal relationship between primary tumor and metastases. Further analysis of single cell genomic and transcriptomic profiles will provide deeper understanding of metastasis in breast cancer. Citation Format: Hakwoo Lee, Farhia Kabeer, Ciara O'Flanagan, Jazmine Brimhall, Justina Biele, Biexi Wang, Teresa Ruiz Algara, So Ra Lee, Daniel Lai, Michael Yuen, Simong Song, Patricia Ye, Jenifer Pham, Richard Moore, Andy J Mungall, Sohrab P Shah, Samuel Aparicio. Single-cell analysis of breast cancer metastasis using patient-derived xenograft model reveals clonal relationship between primary tumor and its metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-05.

Summary Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations., Graphical Abstract, Highlights • Scaled method and resource of > 50K single-cell whole genomes from diverse cell types • Clonal merging can resolve clone specific mutations to single-nucleotide level • Image analysis of single cells permits correlation of morphology and genome features • Clonal replication states and rare aneuploidy patterns of single cells measured, A high-throughput method for amplication-free single-cell whole-genome sequencing can be scaled up to analyze tens of thousands of cells from different tissues and clinical sample types and identifies replication states, aneuploidies, and subclonal mutations.

Measuring gene expression of genomically defined tumour clones at single cell resolution would associate functional consequences to somatic alterations, as a prelude to elucidating pathways driving cell population growth, resistance and relapse. In the absence of scalable methods to simultaneously assay DNA and RNA from the same single cell, independent sampling of cell populations for parallel measurement of single cell DNA and single cell RNA must be computationally mapped for genome-transcriptome association. Here we presentclonealign, a robust statistical framework to assign gene expression states to cancer clones using single-cell RNA-seq and DNA-seq independently sampled from an heterogeneous cancer cell population. We applyclonealignto triple-negative breast cancer patient derived xenografts and high-grade serous ovarian cancer cell lines and discover clone-specific dysregulated biological pathways not visible using either DNA-Seq or RNA-Seq alone.

Prevalence of obesity, an established risk factor for many cancers, has increased dramatically over the past 50 years in the United States and across the globe. Relative to normoweight cancer patients, obese cancer patients often have poorer prognoses, resistance to chemotherapies, and are more likely to develop distant metastases. Recent progress on elucidating the mechanisms underlying the obesity-cancer connection suggests that obesity exerts pleomorphic effects on pathways related to tumor development and progression and, thus, there are multiple opportunities for primary prevention and treatment of obesity-related cancers. Obesity-associated alterations, including systemic metabolism, adipose inflammation, growth factor signaling, and angiogenesis, are emerging as primary drivers of obesity-associated cancer development and progression. These obesity-associated host factors interact with the intrinsic molecular characteristics of cancer cells, facilitating several of the hallmarks of cancer. Each is considered in the context of potential preventive and therapeutic strategies to reduce the burden of obesity-related cancers. In addition, this review focuses on emerging mechanisms behind the obesity-cancer link, as well as relevant dietary interventions, including calorie restriction, intermittent fasting, low-fat diet, and ketogenic diet, that are being implemented in preclinical and clinical trials, with the ultimate goal of reducing incidence and progression of obesity-related cancers.

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 Parkinson's 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.

Obesity is associated with increased risk and poor prognosis of many types of cancers. Several obesity-related host factors involved in systemic metabolism can influence tumor initiation, progression, and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. Such host factors include systemic metabolic regulators including insulin, insulin-like growth factor 1, adipokines, inflammation-related molecules, and steroid hormones, as well as the cellular and structural components of the tumor microenvironment, particularly adipose tissue. These secreted and structural host factors are extrinsic to, and interact with, the intrinsic metabolic characteristics of cancer cells to influence their growth and spread. This review will focus on the interplay of these tumor cell–intrinsic and extrinsic factors in the context of energy balance, with the objective of identifying new intervention targets for preventing obesity-associated cancer.

Mitochondrial activity and metabolic reprogramming influence the phenotype of cancer cells and resistance to targeted therapy. We previously established that an insulin-like growth factor 1 (IGF-1)-inducible mitochondrial UTP carrier (PNC1/SLC25A33) promotes cell growth. This prompted us to investigate whether IGF signaling is essential for mitochondrial maintenance in cancer cells and whether this contributes to therapy resistance. Here we show that IGF-1 stimulates mitochondrial biogenesis in a range of cell lines. In MCF-7 and ZR75.1 breast cancer cells, IGF-1 induces peroxisome proliferator–activated receptor γ coactivator 1β (PGC-1β) and PGC-1α–related coactivator (PRC). Suppression of PGC-1β and PRC with siRNA reverses the effects of IGF-1 and disrupts mitochondrial morphology and membrane potential. IGF-1 also induced expression of the redox regulator nuclear factor-erythroid-derived 2-like 2 (NFE2L2 alias NRF-2). Of note, MCF-7 cells with acquired resistance to an IGF-1 receptor (IGF-1R) tyrosine kinase inhibitor exhibited reduced expression of PGC-1β, PRC, and mitochondrial biogenesis. Interestingly, these cells exhibited mitochondrial dysfunction, indicated by reactive oxygen species expression, reduced expression of the mitophagy mediators BNIP3 and BNIP3L, and impaired mitophagy. In agreement with this, IGF-1 robustly induced BNIP3 accumulation in mitochondria. Other active receptor tyrosine kinases could not compensate for reduced IGF-1R activity in mitochondrial protection, and MCF-7 cells with suppressed IGF-1R activity became highly dependent on glycolysis for survival. We conclude that IGF-1 signaling is essential for sustaining cancer cell viability by stimulating both mitochondrial biogenesis and turnover through BNIP3 induction. This core mitochondrial protective signal is likely to strongly influence responses to therapy and the phenotypic evolution of cancer.

The vast majority of cancer-related deaths are due to metastatic disease, whereby primary tumor cells disseminate and colonize distal sites within the body. Triple negative breast cancer typically displays aberrant Wnt signaling, lacks effective targeted therapies, and compared with other breast cancer subtypes, is more likely to recur and metastasize. We developed a Wnt-driven lung metastasis model of triple negative breast cancer (metM-Wntlung ) through serial passaging of our previously described, nonmetastatic, claudin-low M-Wnt cell line. metM-Wntlung cells displayed characteristics of epithelial-to-mesenchymal transition (e.g., increased invasiveness) with some re-epithealization (e.g., increased adhesion, tight colony formation, increased E-cadherin expression, and decreased Vimentin and Fibronectin expression). When orthotopically transplanted into syngeneic mice, metM-Wntlung cells readily formed tumors and metastasized in vivo, and tumor growth and metastasis were enhanced in obese mice compared with non-obese mice. Gene expression analysis revealed several genes and pathways altered in metM-Wntlung cells compared with M-Wnt cells, including multiple genes associated with epithelial-to-mesenchymal transition, energy metabolism and inflammation. Moreover, obesity caused significant transcriptomic changes, especially in metabolic pathways. Metabolic flux analyses showed greater metabolic plasticity, with heightened mitochondrial and glycolytic energetics in metM-Wntlung cells relative to M-Wnt cells. Similar metabolic profiles were found in a second triple negative breast cancer progression series, M6 and M6C cells. These findings suggest that metabolic reprogramming is a feature of metastatic potential in triple negative breast cancer. Thus, targeting metastases-associated metabolic perturbations may represent a novel strategy for reducing the burden of metastatic triple negative breast cancer, particularly in obese women., Triple negative breast cancer: Obesity and metabolism fuel disease spread Metabolic changes contribute to the metastatic potential of triple negative breast cancer (TNBC), a mouse study shows. Stephen Hursting and colleagues from the University of North Carolina at Chapel Hill, USA, established metastatic mouse TNBC cells driven by Wnt-1, a signaling protein that’s highly active in this aggressive subtype of breast cancer. In a lab dish, these cells showed signs of increased invasiveness; and when transplanted into mice, the cells readily formed tumors that metastasized to the lungs. Obese mice experienced more aggressive tumor growth and spread than normal-weight animals. Gene expression analyses revealed that TNBC cells with metastatic potential have an energetic leg-up over their non-metastatic counterparts in the face of obesity-induced metabolic changes, suggesting that targeting metabolic perturbations could help reduce the burden of metastatic TNBC, particularly for obese women.

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on 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 AKT or MAPK-signaling pathways in different cells. The IGF-1R, β1 Integrin, FER, and its substrate cortactin were all observed to co-locate 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 anaplastic lymphoma kinase inhibitor (AP26113) 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.

Single cell RNA sequencing (scRNAseq) is a powerful tool, particularly for studying complex biological systems, such as tumor heterogeneity and the tumor microenvironment, which may not be resolved by sequencing of bulk material. Nonetheless, it is not without limitations, which include the technical challenges of generating a high quality single cell suspension. Dissociation of tissue to single cell suspension requires mechanical and enzymatic disruption, and the effect of these methods on gene expression or cellular population bias has not been established. In this study, we examined the effects of enzymatic dissociation on cell population capture and transcriptional changes at single cell resolution in breast and ovarian cancer patient samples, patient-derived breast cancer xenografts and cultured cell lines. scRNAseq data showed that enzymatic dissociation of tissues at 37oC with collagenase resulted in significant induction of heat shock, stress and immediate response genes, which was conserved across all tissues. This gene expression induction was not observed when tissues were dissociated at 6oC with a protease derived from a Himalayan glacier soil bacterium. Moreover, dissociation of patient tumors at low temperature enhanced the abundance of rare cell populations, including B-cells, T-Cells and cytotoxic T-cells, which were significantly depleted following dissociation at 37oC. These biases resulting from standard sample preparation methods could significantly affect biological interpretation of scRNAseq data, and can be minimized by dissociation of tissues at low temperature. Note: This abstract was not presented at the meeting. Citation Format: Ciara H. O'Flanagan, Kieran R. Campbell, Farhia Kabeer, Allen Zhang, Jamie Lim, Sohrab P. Shah, Samuel Aparicio. Temperature-dependent transcription artifacts and cell population biases in scRNAseq data are minimized by tissue dissociation at low temperatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4706.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers worldwide, with a 5- year survival rate of less than 5 percent. KRAS-driven PDACs often exhibit increased dependence on autophagy, a process by which cells degrade internal components to mobilize energy stores. When faced with cellular stress, such as nutrient deprivation, tumor growth may initially slow in response to decreasing supplies of growth factors and cytokines. Under these conditions, the induction of autophagy can enable continued tumor growth even in the presence of nutrient stress. Therefore, we posited that nutrient restriction in combination with autophagy inhibition would synergistically disrupt aberrant metabolic pathways and more effectively stunt PDAC tumor progression. To determine the impact of transient autophagy inhibition, cells were treated with 10 μM of the pharmaceutical agent chloroquine (CQ) for 48 hours. To modulate growth factor/cytokine signaling as well as nutrient sensing signaling serum and glucose restriction were used in vitro. Next, permanent autophagy ablation was achieved through CRISPR/Cas9 technology, autophagy related 5 (Atg5) was deleted in Panc02 cells generating an autophagy-deficient (Atg-/-) PDAC cell line. Growth kinetics were determined in a range of culture matrixes to determine the proliferation rate, invasiveness, and anchorage independence of cells. The combination of CQ and nutrient stress elicited an additive effect on cellular proliferation in both murine and human-derived KRAS mutant PDAC cells (Panc02 and MIA PaCa-2 respectively). In contrast, Atg5-/- Panc02 cells did not display any alteration in cellular proliferation in vitro. Atg5 deletion resulted in decreased expression of mesenchymal markers N-Cadherin and Snail as well as decreased invasiveness and anchorage independent colony formation. Upon intrapancreatic injection in a syngeneic model, Atg-/- cells exhibited reduced tumor growth relative to control Atg5+/+cells, indicating the compensatory mechanisms effective in vitro failed to protect these cells in vivo. Thus, our findings reveal a potential synergism between autophagy inhibition and nutrient stress in PDAC. Citation Format: Jane B. Pearce, Ciara H. O'Flanagan, Stephen D. Hursting. Transient versus permanent autophagy inhibition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4265.

Triple-negative breast cancers (TNBC) are an aggressive BC subset currently lacking targeted therapies. TNBCs exhibit aberrant fuel metabolism, including increased flux through glycolysis and the pentose phosphate pathway, increased lactate production (Warburg metabolism), and elevated dependence on glutamine (glutaminolysis). While dietary interventions such as caloric restriction, intermittent fasting, or ketogenic diets have demonstrated benefits (e.g, reduced tumor incidence, progression, and sensitization to some chemotherapeutic agents) in preclinical BC models, translation to humans has proven challenging, and concerns remain regarding tolerance and long-term safety of extreme diets in cancer patients. We hypothesized that targeted disruption of select metabolic pathways could recapitulate the beneficial effects of the aforementioned dietary interventions and improve the efficacy of carboplatin, part of first-line chemotherapy for primary and metastatic TNBCs. Moreover, we posited that blocking autophagy would further enhance the anticancer effects of metabolic reprogramming interventions (MRIs) alone and in combination with carboplatin. To test these hypotheses, we are using a high-throughput screening approach employing several MRIs with or without the addition of chloroquine and/or carboplatin in vitro. Serum starvation (an in vitro model of caloric restriction), rapamycin, BMS-754807 (a dual IGF-1R/insulin receptor), and nicotinamide significantly reduced cell viability (assessed by MTT assay) and clonogenicity (assessed by colony formation assay) of M-Wnt and M6 cells, both well-characterized TNBC cell lines derived from mammary tumors of MMTV-Wnt-1 and C3-TAg transgenic mice, respectively. The growth suppressive effects of these MRIs were further enhanced when combined with autophagy inhibition via chloroquine (CQ). Treatment of M-Wnt and M6 cells with CQ (20-80 µM) also increased in vitro cytotoxicity of carboplatin. Ongoing analyses and planned in vivo studies will confirm whether co-treatment with MRIs and/or CQ are an effective strategy for increasing the efficacy of carboplatin in TNBC cells. Citation Format: Alyssa J. Cozzo, Michael F. Coleman, Ciara H. O'Flanagan, Jane B. Pearce, Magdalena A. Rainey, Stephen D. Hursting. Separate and combined effects of metabolic reprogramming interventions, autophagy inhibition, and carboplatin on murine triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5278.

// Ciara H. O’Flanagan 1, * , Sandra O’Shea 1, * , Amy Lyons 1 , Fionola M. Fogarty 1 , Nuala McCabe 2 , Richard D. Kennedy 2, 3 , Rosemary O’Connor 1 1 School of Biochemistry and Cell Biology, University College Cork, Cork, Ireland 2 Almac Diagnostics, Craigavon, Northern Ireland, UK 3 Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, Northern Ireland, UK * Joint authorship Correspondence to: Rosemary O’Connor, email: r.oconnor@ucc.ie Keywords: IGF-1R, DNA damage, breast cancer, TKI Received: April 11, 2016 Accepted: July 10, 2016 Published: July 27, 2016 ABSTRACT The complexity of the IGF-1 signalling axis is clearly a roadblock in targeting this receptor in cancer therapy. Here, we sought to identify mediators of resistance, and potential co-targets for IGF-1R inhibition. By using an siRNA functional screen with the IGF-1R tyrosine kinase inhibitor (TKI) BMS-754807 in MCF-7 cells we identified several genes encoding components of the DNA damage response (DDR) pathways as mediators of resistance to IGF-1R kinase inhibition. These included ATM and Ataxia Telangiectasia and RAD3-related kinase (ATR). We also observed a clear induction of DDR in cells that were exposed to IGF-1R TKIs (BMS-754807 and OSI-906) as indicated by accumulation of γ-H2AX, and phosphorylated Chk1. Combination of the IGF-1R/IR TKIs with an ATR kinase inhibitor VE-821 resulted in additive to synergistic cytotoxicity compared to either drug alone. In MCF-7 cells with stably acquired resistance to the IGF-1R TKI (MCF-7-R), DNA damage was also observed, and again, dual inhibition of the ATR kinase and IGF-1R/IR kinase resulted in synergistic cytotoxicity. Interestingly, dual inhibition of ATR and IGF-1R was more effective in MCF-7-R cells than parental cells. IGF-1R TKIs also potentiated the effects of cisplatin in a panel of breast cancer cell lines. Overall, our findings identify induction of DDR by IGF-1R kinase inhibition as a rationale for co-targeting the IGF-1R with ATR kinase inhibitors or cisplatin, particularly in cells with acquired resistance to TKIs.

Breast cancer (BC) is the most common noncutaneous cancer among US women, and ~20% of these cancers overproduce the growth-promoting tyrosine kinase receptor, HER2. Although there are targeted treatments available for HER2-overexpressing BC, there remains an urgent need to identify new treatment strategies since this disease is associated with poor prognosis, resistance to therapy, and high risk of recurrence. We have established that calorie restriction (CR; 30% reduction in total energy relative to ad libitum-fed controls) has significant tumor suppressive effects across all breast cancer subtypes and has potential to be utilized as an adjunctive therapy. This study investigates the mechanisms by which CR decreases HER2-overexpressing BC progression. To mimic CR in vitro, murine MMTV-neu cells that overexpress neu, the rodent homolog of HER2, were treated with media containing reduced serum (1%), reduced glucose (1mM), or reduced serum and reduced glucose (1%/1mM) compared to control media (10% serum, 25mM glucose). MTT assays demonstrated that both serum restricted medias (1% and 1%/1mM) significantly decreased cellular viability (p Citation Format: Magdalena A. Rainey, Laura A. Smith, Ciara H. O'Flanagan, Stephen D. Hursting. Short-term calorie restriction alters expression of tumor suppressor p21 in HER2-overexpressing breast cancer [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 2396.

Purpose: Approximately 20% of breast cancers (BC) among US women overexpress HER2, an oncoprotein that stimulates increased cellular proliferation and survival. HER2 acts through two major signaling pathways, PI3K/Akt and Raf/MAPK, and its overexpression is associated with aggressive disease, resistance to therapy, and poor prognosis. Our laboratory has previously demonstrated in preclinical models that calorie restriction (CR) modulates HER2 BC pathogenesis, decreasing incidence and increasing latency in transgenic MMTV-HER2/neu mice. Our current study aims to determine the impact of CR on HER2 signaling and cancer cell phenotypes, including proliferation, cell cycle progression, and apoptosis. Methods: In vitro experiments were performed using a BC cell line derived from the MMTV-HER2/neu mouse model. To mimic CR, cells were treated with nutrient restricted media containing either reduced glucose (1mM), reduced serum (1%) or both (1mM/1%). The impact of CR on proliferation and survival was measured using growth curve experiments, and flow cytometric analysis of cell cycle and apoptosis. HER2 signaling proteins were assessed by western blotting. Results: Relative to cells grown in control media (25mM glucose/10% BCS): a) cells grown in glucose restricted (1mM) + serum restricted (1%) media grew 87% slower (p Citation Format: Laura A. Smith, Magdalena A. Rainey, Nishita T. Sheth, Ciara H. O'Flanagan, Laura W. Bowers, Stephen D. Hursting. Calorie restriction reduces PI3K/Akt signaling and tumorigenic potential in HER2-overexpressing breast cancer [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 379.

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 O'Shea, Michael Coleman, Ciara O'Flanagan, Barbara Addario, Nuala McCabe, Richard Kennedy, Rosemary O'Connor. 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.

Pancreatic cancer is the 4th most fatal cancer in US men and women, with 5-year survival rates under 5%. Obesity is one risk factor known to increase pancreatic cancer risk by facilitating tumorigenesis and metabolic processes associated with pancreatic tumor growth and progression. Autophagy is the catabolic process of recycling cellular organelles to provide energy to the cell and is often induced when cells become stressed. Autophagy plays a critical role in cancer growth and metastasis, and in many cancers autophagy inhibition decreases the size of tumors in in vivo models. Our current studies investigate the link between the autophagy inhibition and the invasiveness of pancreatic cancer. To understand the role of autophagy on invasion, EMT and 3D growth in pancreatic cancer, Kras-driven Panc02 mouse pancreatic cancer cells were transduced using CRISPR/Cas9 to cleave the gene encoding autophagy related protein 5 (Atg5). Atg5 deletion resulted in striking morphological alterations, including a more epithelial-like appearance. In addition, Atg5 deletion resulted in increased expression of the epithelial marker E-Cadherin, concurrent with decreased expression of mesenchymal markers N-Cadherin and Snail. Furthermore, Atg5 deletion resulted in significant decreased invasiveness and ability to form colonies under anchorage-independent conditions, with no significant difference in 2D cell growth. Orthotopic, intrapancreatic injection of these cells into C57BL/6 mice resulted in reduced tumor take and growth. Taken together, these results for the first time show that autophagy inhibition results in reduced EMT and 3D growth in pancreatic adenocarcinoma and highlight autophagy's role in pancreatic adenocarcinoma progression. Citation Format: Jane B. Pearce, Ciara H. O'Flanagan, Stephen D. Hursting. Autophagy inhibition reverses EMT and reduces 3D growth of pancreatic adenocarcinoma cells in vitro and in vivo [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 1336.

Calorie restriction (CR) is one of the most effective dietary interventions for reducing cancer risk and progression. However, maintaining a dramatically reduced calorie intake over an extended time period is very challenging for most individuals. Thus a major effort is under way to identify mechanism-based pharmacologic approaches that mimic the anticancer effects of CR without the requirement of reduced calorie intake. We have established that CR exerts antitumor effects at least in part through altered metabolism, reduced circulating growth factors (such as insulin and IGF-1) and reduced signaling downstream of the insulin receptor and IGF-1R along the phosphoinositide 3-kinase/mTOR axis. We have previously shown CR reduced growth of KRAS-driven tumors in vivo, and the antitumorigenic effect was enhanced when combined with autophagy inhibition. Here we evaluated the effects of in vitro CR via glucose and serum modulation, as well as separate and combined effects of CR mimetics and autophagy inhibition on the KRAS-driven murine pancreatic adenocarcinoma (PDAC) cell line, FC1242. Glucose modulation alone via glucose restriction, metformin or AICAR treatment showed no effect on clonogenic growth of FC1242. In contrast, both cell viability and clonogenicity were significantly reduced by serum starvation, rapamycin treatment, IGF-1R/insulin receptor inhibition by BMS-754807, and sirtuin inhibition with nicotinamide, suggesting these cells are dependent on mTOR/IGF-1 signaling. The growth-suppressive effects of these CR mimetics were enhanced when combined with autophagy inhibition via chloroquine or verteporfin. Together, the study indicated the targeting of mTOR or IGF-1 signaling may most closely mimic the growth-suppressive effects of CR, and that co-treatment of these CR mimetics with autophagy inhibition may be an effective strategy for PDAC anticancer therapy. Citation Format: Xuewen Chen, Ciara H. O'Flanagan, Michael Coleman, Channing J. Der, Stephen D. Hursting. Separate and combined effects of caloric restriction mimetics and autophagy inhibition on KRAS-driven pancreatic adenocarcinoma [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 3508.

Numerous epidemiological and pre-clinical studies have demonstrated that the insulin/insulin-like growth factor (IGF) system plays a key role in the development and progression of several types of cancer. Insulin/IGF signaling, in cooperation with chronic low-grade inflammation, is also an important contributor to the cancer-promoting effects of obesity. However, clinical trials for drugs targeting different components of this system have produced largely disappointing results, possibly due to the lack of predictive biomarker use and problems with the design of combination therapy regimens. With careful attention to the identification of likely patient responders and optimal drug combinations, the outcome of future trials may be improved. Given that insulin/IGF signaling is known to contribute to obesity-associated cancer, further investigation regarding the efficacy of drugs targeting this system and its downstream effectors in the obese patient population is warranted.

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. ispartof: Oncogene vol:34 issue:11 pages:1363-1374 ispartof: location:England status: published

Cancer and neurodegeneration are two age-related diseases that arise from aberrant signaling in similar cellular systems, those that balance survival and death. Thus, deregulated molecular processes such as DNA damage repair, intracellular energy balance, and key signal transduction systems, including the PI3-kinase/Akt axis can promote tumorigenesis and induce neurodegeneration [1]. Epidemiological studies support this cross-talk between cancer and neurodegeneration, indicating a reduced risk of certain cancers in patients diagnosed with neurodegenerative diseases such as Parkinson's disease (PD) [2]. In addition, several of the genes discovered to cause inherited PD, including PTEN induced putative kinase 1 (PINK1) have been described to have oncogenic or tumor suppressor properties [3]. In a recent study we focused on the function of PINK1 in cancer cell biology, and discovered a novel function for PINK1 as a positive regulator of cell cycle progression that can promote cancer-associated phenotypes [4]. PINK1 is ubiquitously expressed and was named due to induction by the tumor suppressor PTEN in cancer cells, drawing attention to its putative role in cancer from the first instance. Several mechanistic links between PINK1, PTEN and the PI3-kinase/Akt signaling axis that PTEN inhibits were subsequently highlighted, indicating PINK1 is both regulated by and regulates PI3-kinase/Akt signaling [5]. Interlinked with this, in an as yet undefined manner, PINK1 is best described as a major mitochondrial quality control protein, rudimentary to cell survival due to its regulatory role in the triad of mitochondrial fission, fusion and mitophagy as well as mitochondrial bioenergetics. Although somewhat understudied, the cell cycle and mitochondrial quality control are intrinsically coupled [6]. Mitochondria must divide and undergo fission during mitosis to allow equal distribution of mitochondria to daughter cells, also permitting clearance of damaged mitochondria via mitophagy. Conversely, mitochondrial fusion occurs during the transition from mitosis to G1 following cytokinesis, and can promote stress resistance and cell cycle exit in G0. Our findings show for the first time that regulation of mitochondrial fission to fusion transitions by PINK1 is critical for cell cycle progression at G2/M and G0/G1 checkpoints necessary for cell division, growth and stress resistance, in particular in cancer biology. In line with this, PINK1 deletion reduced proliferation, colony formation, migration and invasive potential in several cell model systems. In further detail, PINK1-deficiency induced multinucleation and cell cycle arrest during G2/M and resulted in a reduced ability to exit the cell cycle following serum withdrawal. This was PINK1 kinase dependent and rescued by re-introduction of human PINK1. The cell cycle changes induced by PINK1 deletion where mechanistically linked to excessive mitochondrial fission, and increased expression and activation of the master mitochondrial fission protein dynamin-related protein 1 (Drp1). siRNA knockdown of Drp1 and restoration of mitochondrial fusion in PINK1-deficient cells caused a reduced multi-nucleation. Together this indicates that mitotic arrest with an inability to complete cytokinesis in cells without PINK1 is due to excessive mitochondrial fission, and an inability to induce fusion following nuclear separation and prior to cytokinesis. Significant cell cycle marker changes were co-existent with this defect including failure to increase cyclin D1, indicative of mitotic arrest induced by PINK1 deletion. PINK1 has been previously highlighted as a potential target for cancer therapy and been shown to sensitize cancer cells to DNA damaging agents and chemotherapeutic drugs [7]. Our findings show that PINK1 inhibition constrains proliferation, halting the cell just before division, the point at which many of these agents target. PINK1 may therefore be a direct target to block the cell cycle in cancer or for combination therapies to ‘prime’ cancer cells for treatment with other mitosis-targeting drugs. Conversely, the inability of cells to effectively divide in the absence of PINK1 has the potential to increased chromosomal aberrations, genetic instability and aneuploidy that could lead to cancer in some cell types. This context dependent pro- and antitumorigenic properties depending on cell type, is emerging for many genes with oncogenic potential. The significance of PINK1's involvement in cell cycle regulation is important not only in cancer whereby cells are continually dividing, but also in neuronal biology, as abortive cell cycle re-entry in terminally differentiated, post-mitotic neurons has been suggested to be a key mechanism in neurodegenerative diseases [3]. While much is known about the function of PINK1 in mitochondrial biology and also to a lesser but growing extent, in PI3-kinase/Akt signalling, the exact mechanism through which loss of function of PINK1 causes PD is still unknown. This new function of PINK1 as a regulator of the cell cycle draws attention to the function of PINK1, via mitochondrial quality control, in both cell division, and cell differentiation programs, that underlie cancer and adult neuronal phenotypes. These findings therefore add another piece towards solving the larger puzzle of PINK1 function in neuronal systems and highlight the potential of PINK1 as a target in future anti-cancer therapies.

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. O'Flanagan, 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

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. O'Flanagan, 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

Epithelial-to-mesenchymal transition (EMT), the process through which epithelial cells gain mesenchymal characteristics including reduced adhesion and enhanced invasion, is a critical step in progression of cancer cells toward metastasis. However, the molecular mechanisms governing this process remain poorly understood. Furthermore, though metastasis is the cause of 90% of cancer deaths, no targeted therapy is currently available for metastatic disease. Metabolic reprogramming is a key feature of most cancer cells, with oxidative phosphorylation often being switched off in favor of glycolytic and synthetic pathways. Autophagy is a catabolic process in which protein complexes, damaged organelles and other macromolecules are lysosomally degraded. Autophagy can promote cancer cell survival, providing protein, lipid, nucleic acid and membrane precursors as well as auxiliary energy during periods of energy stress. The role of metabolism in EMT and metastasis has not been well studied. We hypothesized that the transition to metastasis involves metabolic alterations, including activation of autophagy. Previously, we derived (from a spontaneous mammary tumor in an MMTV-Wnt transgenic mouse) epithelial-like (E-Wnt) and mesenchymal-like (M-Wnt) cell lines, which recapitulate basal-like and claudin-low and breast tumors, respectively. Here, a metastatic derivative of M-Wnt cells was generated from lung metastases following serial passages in a severe combined immunodeficient (SCID) mouse. metM-Wnt cells were more proliferative, invasive and formed more colonies in soft agar than their parental M-Wnt cell line. Tail vein injection or mammary fat pad orthotopic implantation of metM-Wnt cells resulted in metastatic tumor formation in either the lungs or liver as detected by IVIS imaging and histological analysis. metM-Wnt cells displayed higher rates of glycolysis and oxidative phosphorylation, indicating that these cells are highly energetic. Furthermore, M-Wnt and metM-Wnt cells and tumors were found to have increased autophagic flux, as measured by LC3B expression and cleavage compared to E-Wnt cells and tumors. metM-Wnt cells were more sensitive to autophagy inhibition (either via chloroquine treatment or knockout of Atg5, a key component of the autophagic machinery) than M-Wnt or E-Wnt cells. Conversely, metM-Wnt cells were resistant to treatment with rapamycin, concomitant with sustained activation of mTOR, which controls many metabolic pathways including autophagy and protein synthesis. These results indicate that metabolic alteration is a feature of EMT in claudin-low breast cancer, including increased glycolysis, oxidative phosphorylation and autophagy. Furthermore, metastatic breast cancer cells may be more reliant on autophagy than non-metastatic cells, and autophagy may therefore be a therapeutic target in which to treat metastatic breast cancers. Citation Format: Ciara H. O’Flanagan, Emily L. Rossi, Stephen D. Hursting. Autophagy forms part of a metabolic switch during epithelial-to-mesenchymal transition and metastasis in a murine claudin-low breast cancer model. [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 1020.

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 O'Flanagan, Deirdre A. Buckley, Patrick A. Kiely, Rosemary O'Connor. 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

Calorie restriction (CR) prevents obesity and exerts anticancer effects in many preclinical models. CR is also increasingly being used in cancer patients as a sensitizing strategy prior to chemotherapy regimens. While the beneficial effects of CR are widely accepted, the mechanisms through which CR affects tumor growth are incompletely understood. In many cell types, CR and other nutrient stressors can induce autophagy, which provides energy and metabolic substrates critical for cancer cell survival. We hypothesized that limiting extracellular and intracellular substrate availability by combining CR with autophagy inhibition would reduce tumor growth more effectively than either treatment alone. A 30 % CR diet, relative to control diet, in nude mice resulted in significant decreases in body fat, blood glucose, and serum insulin, insulin-like growth factor-1, and leptin levels concurrent with increased adiponectin levels. In a xenograft model in nude mice involving H-RasG12V-transformed immortal baby mouse kidney epithelial cells with (Atg5 +/+ ) and without (Atg5 −/−) autophagic capacity, the CR diet (relative to control diet) genetically induced autophagy inhibition and their combination, each reduced tumor development and growth. Final tumor volume was greatest for Atg5 +/+ tumors in control-fed mice, intermediate for Atg5 +/+ tumors in CR-fed mice and Atg5 −/− tumors in control-fed mice, and lowest for Atg5 −/− tumors in CR mice. In Atg5 +/+ tumors, autophagic flux was increased in CR-fed relative to control-fed mice, suggesting that the prosurvival effects of autophagy induction may mitigate the tumor suppressive effects of CR. Metabolomic analyses of CR-fed, relative to control-fed, nude mice showed significant decreases in circulating glucose and amino acids and significant increases in ketones, indicating CR induced negative energy balance. Combining glucose deprivation with autophagy deficiency in Atg5 −/− cells resulted in significantly reduced in vitro colony formation relative to glucose deprivation or autophagy deficiency alone. Combined restriction of extracellular (via CR in vivo or glucose deprivation in vitro) and intracellular (via autophagy inhibition) sources of energy and nutrients suppresses Ras-driven tumor growth more effectively than either CR or autophagy deficiency alone. Interventions targeting both systemic energy balance and tumor-cell intrinsic autophagy may represent a novel and effective anticancer strategy.