Zeb1 mediates EMT/plasticity-associated ferroptosis sensitivity in cancer cells by regulating lipogenic enzyme expression and phospholipid composition.

Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFß stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.

Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

ATF3 characterizes aggressive drug-tolerant persister cells in HGSOC.

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients.

Indigenizing and Ruralizing NCI Screen to Save Program: Resources, Optimizing Outreach, Teaching, Science (ROOTS).

Colorectal cancer (CRC) is a complex health disparity in many Indigenous and rural populations. While it affects anyone regardless of race, age, gender, or other common differences among people, Indigenous and rural populations are at a higher risk of dying from colorectal cancer. An NCI Screen to Save (S2S) program was culturally tailored to promote awareness and knowledge of colorectal cancer and screening in both Indigenous and rural communities across a sector in Northeastern USA. Indigenous and rural community outreach teams at an NCI-designated cancer center partnered with a community advisory board to provide an indigenized/ruralized version of the NCI Screen to Save program delivered to both Indigenous and rural/suburban communities. In total, n = 79 pre/post surveys were obtained from n = 82 participants, who had an average age of 49 years. Findings demonstrated that Indigenous/rural participants in both off-territory/non-reservation communities and a tribal community that received a culturally tailored version of NCI's S2S program were able to identify both smoking and tobacco use along with lack of physical activity as risk factors for colorectal cancer. Post-intervention, participants reported being more likely to increase physical activity. Most importantly, participants said they would be more likely to be screened for colorectal cancer along with their family and friends based on their cancer screening experiences. Culturally tailored CRC messaging is an effective means for increasing screening intentions and decreasing cancer health disparities among both indigenous and rural populations. Future research should include the relationship of diet to obesity-related cancers, greater integration of Indigenous-rural patient navigation programs, creation of more information on genetic screening, and quality improvement to service translational science initiatives.

MMD collaborates with ACSL4 and MBOAT7 to promote polyunsaturated phosphatidylinositol remodeling and susceptibility to ferroptosis.

Ferroptosis is a form of regulated cell death with roles in degenerative diseases and cancer. Excessive iron-catalyzed peroxidation of membrane phospholipids, especially those containing the polyunsaturated fatty acid arachidonic acid (AA), is central in driving ferroptosis. Here, we reveal that an understudied Golgi-resident scaffold protein, MMD, promotes susceptibility to ferroptosis in ovarian and renal carcinoma cells in an ACSL4- and MBOAT7-dependent manner. Mechanistically, MMD physically interacts with both ACSL4 and MBOAT7, two enzymes that catalyze sequential steps to incorporate AA in phosphatidylinositol (PI) lipids. Thus, MMD increases the flux of AA into PI, resulting in heightened cellular levels of AA-PI and other AA-containing phospholipid species. This molecular mechanism points to a pro-ferroptotic role for MBOAT7 and AA-PI, with potential therapeutic implications, and reveals that MMD is an important regulator of cellular lipid metabolism.

Sweet Beverages and Cancer: A Scoping Review of Quantitative Studies.

We conducted a scoping review of sweet beverages (SB) and cancer outcomes to ascertain SB's relationship with cancer by SB type and cancer type. We used the PRISMA Scoping Review Guidelines to review quantitative studies of SB and cancer. Eligible studies included articles reporting a quantitative association between SB intake and a cancer-related health outcome in humans, including adiposity-related versus non-adiposity-related cancers. Studies included analyses not confounded by artificial sweeteners. SB was defined as beverages with added sugars, 100% fruit juices, or fruit drinks that were not 100% fruit juice. We used a data-charting form to extract study characteristics and results.A total of 38 were included. The sample consisted predominately of adults from European countries outside of the United States or predominately White samples in the United States. Across all conceptualizations of SB, a greater proportion of studies examining carbonated drinks reported SB's relationship with poorer cancer outcomes, which was exacerbated in adiposity-related cancers.The composition of different types of SB (e.g., high fructose corn syrup, natural fructose) as they relate to cancer is important. Studies including more diverse populations that bear a disproportionate burden of both SB intake and cancer are needed. PREVENTION RELEVANCE: Different sugars in SB may impact cancer differently. Compared with SB made with other types of sugar, drinks made with man-made fructose (carbonated drinks) had poorer cancer outcomes, especially in cancers impacted by obesity. Understanding how different SB affect cancer would help us target which SB to avoid.

Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Views on clinical trial recruitment, biospecimen collection, and cancer research: population science from landscapes of the Haudenosaunee (People of the Longhouse).

Biomedical research in culturally distinct communities is often a challenge. Potential barriers to participation occur because science is presented in a format that lacks cultural acknowledgement. Investigations may also fail to showcase beneficial relevance to the communities or include them in true partnership. The history of biomedical research within Native American societies has been complicated by these issues. Historical trauma among many Native groups sometimes transcends into contemporary challenges in both recruitment to and participation particularly in biobanking research. The participants for this study included members of the Haudenosaunee, the People of the Longhouse. Native Americans, including the Haudenosaunee, endure some of the worst health disparities in the country. These include high rates of cancer, obesity, and diabetes which may be linked at least partially to genetic predisposition. Results from a Haudenosaunee urban population shared response on ways to improve recruitment strategies for biospecimen, cancer, and other health-related clinical trials. Mixed methods approaches were used, and community responses indicated the importance of creating trust through respectful partnership; promoting culturally appropriate recruitment materials; the need for a greater understanding of consenting and signature processes; the necessity for concise summary sheets; and a desire to have information that community member understand. Discussion items also include international Indigenous perspectives to biobanking and genetic-related health disparity research.

Mortality disparities: A comparison with the Haudenosaunee in New York State.

Identifying health status and disparities for Indigenous populations is the first logical step toward better health. We compare the mortality profile of the American Indian and Alaska Native (AI/AN) population with that of non-Hispanic whites in the Haudenosaunee Nations in New York State, the Indian Health Service (IHS) East region (Nashville Area) and the United States. Data from the linkage of IHS registration records with decedents from the National Death Index (1990-2009) were used to identify AI/AN deaths misclassified as non-AI/AN. Analyses were limited to persons of non-Hispanic origin. We analyzed trends for 1990-2009 and compared AI/AN and white persons in the Haudenosaunee Nations in New York State, IHS East region and the United States. All-cause death rates over the past two decades for Haudenosaunee men declined at a greater percentage per year than for AI/AN men in the East region and United States. This decrease was not observed for Haudenosaunee women with all-cause death rates appearing to be stable over the past two decades. Haudenosaunee all-cause death rates were 16% greater than that for whites in the Haudenosaunee Nations. The most prominent disparities between Haudenosaunee and whites are concentrated in the 25-44 year age group (Risk Ratio=1.85). Chronic liver disease, diabetes, unintentional injury, and kidney disease death rates were higher in Haudenosaunee than in whites in the Haudenosaunee Nations. The Haudenosaunee cancer death rate (180.8 per 100,000) was higher than that reported for AI/AN in the East (161.5 per 100,000).Haudenosaunee experienced higher rates for the majority of the leading causes of death than East AI/AN. These results highlight the importance of Haudenosaunee-specific data to target prevention efforts to address health disparities and inequalities in health.

Pentraxin-3 is a PI3K signaling target that promotes stem cell-like traits in basal-like breast cancers.

Basal-like breast cancers (BLBCs) exhibit hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway because of the frequent mutational activation of the PIK3CA catalytic subunit and the genetic loss of its negative regulators PTEN (phosphatase and tensin homolog) and INPP4B (inositol polyphosphate-4-phosphatase type II). However, PI3K inhibitors have had limited clinical efficacy in BLBC management because of compensatory amplification of PI3K downstream signaling loops. Therefore, identification of critical PI3K mediators is paramount to the development of effective BLBC therapeutics. Using transcriptomic analysis of activated PIK3CA-expressing BLBC cells, we identified the gene encoding the humoral pattern recognition molecule pentraxin-3 (PTX3) as a critical target of oncogenic PI3K signaling. We found that PTX3 abundance is stimulated, in part, through AKT- and nuclear factor κB (NF-κB)-dependent pathways and that presence of PTX3 is necessary for PI3K-induced stem cell-like traits. We further showed that PTX3 expression is greater in tumor samples from patients with BLBC and that it is prognostic of poor patient survival. Our results thus reveal PTX3 as a newly identified PI3K-regulated biomarker and a potential therapeutic target in BLBC.

Aspirin Suppresses Growth in PI3K-Mutant Breast Cancer by Activating AMPK and Inhibiting mTORC1 Signaling.

Despite the high incidence of oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of PI3K, PI3K inhibitors have yielded little clinical benefit for breast cancer patients. Recent epidemiologic studies have suggested a therapeutic benefit from aspirin intake in cancers harboring oncogenic PIK3CA Here, we show that mutant PIK3CA-expressing breast cancer cells have greater sensitivity to aspirin-mediated growth suppression than their wild-type counterparts. Aspirin decreased viability and anchorage-independent growth of mutant PIK3CA breast cancer cells independently of its effects on COX-2 and NF-κB. We ascribed the effects of aspirin to AMP-activated protein kinase (AMPK) activation, mTORC1 inhibition, and autophagy induction. In vivo, oncogenic PIK3CA-driven mouse mammary tumors treated daily with aspirin resulted in decreased tumor growth kinetics, whereas combination therapy of aspirin and a PI3K inhibitor further attenuated tumor growth. Our study supports the evaluation of aspirin and PI3K pathway inhibitors as a combination therapy for targeting breast cancer. Cancer Res; 77(3); 790-801. ©2016 AACR.

LINC00520 is induced by Src, STAT3, and PI3K and plays a functional role in breast cancer.

Long non-coding RNAs (lncRNAs) have been implicated in normal cellular homeostasis as well as pathophysiological conditions, including cancer. Here we performed global gene expression profiling of mammary epithelial cells transformed by oncogenic v-Src, and identified a large subset of uncharacterized lncRNAs potentially involved in breast cancer development. Specifically, our analysis revealed a novel lncRNA, LINC00520 that is upregulated upon ectopic expression of oncogenic v-Src, in a manner that is dependent on the transcription factor STAT3. Similarly, LINC00520 is also increased in mammary epithelial cells transformed by oncogenic PI3K and its expression is decreased upon knockdown of mutant PIK3CA. Additional expression profiling highlight that LINC00520 is elevated in a subset of human breast carcinomas, with preferential enrichment in the basal-like molecular subtype. ShRNA-mediated depletion of LINC00520 results in decreased cell migration and loss of invasive structures in 3D. RNA sequencing analysis uncovers several genes that are differentially expressed upon ectopic expression of LINC00520, a significant subset of which are also induced in v-Src-transformed MCF10A cells. Together, these findings characterize LINC00520 as a lncRNA that is regulated by oncogenic Src, PIK3CA and STAT3, and which may contribute to the molecular etiology of breast cancer.

Developing an obesity-cancer intervention for workplaces: Indigenous, Native American, Maori and other minority occupational settings.

There is growing evidence for links between obesity and certain types of cancer. Studies done within Native American, Maori, and other Indigenous populations suggest the need to promote healthier lifestyles, including the maintenance of optimal body weight through nutrition and physical activity, to lower the risk factors of obesity-related cancers. What is missing is a program that combines culturally attuned workplace interventions that deal with obesity reduction as it relates to cancer prevention. The main purpose of this project was to discuss the process of developing an employee assistance program module to reduce the risk for obesity-related cancers. Expert curriculum developers specialising in workplace disease management assisted with the creation of a unique obesity and cancer prevention program. Several national leaders in Indigenous and minority health were consulted for feedback. The completed intervention included a six-session model with cultural features wrapped around topics of obesity-related cancer warning signs, diet and physical activity guidance, stress management, goal-setting, and resource linkage. A Native American workplace was selected for feasibility and pilot testing. Preliminary results are also discussed. Ultimately, this paper presents a novel intervention approach to address health issues for Native Americans, with indicators for use in other Indigenous populations globally.

NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer.

NFAT transcription factors are key regulators of gene expression in immune cells. In addition, NFAT1-induced genes play diverse roles in mediating the progression of various solid tumors. Here we show that NFAT1 induces the expression of the IL8 gene by binding to its promoter and leading to IL8 secretion. Thapsigargin stimulation of breast cancer cells induces IL8 expression in an NFAT-dependent manner. Moreover, we show that NFAT1-mediated IL8 production promotes the migration of primary human neutrophils in vitro and also promotes neutrophil infiltration in tumor xenografts. Furthermore, expression of active NFAT1 effectively suppresses the growth of nascent and established tumors by a non cell-autonomous mechanism. Evaluation of breast tumor tissue reveals that while the levels of NFAT1 are similar in tumor cells and normal breast epithelium, cells in the tumor stroma express higher levels of NFAT1 compared to normal stroma. Elevated levels of NFAT1 also correlate with increased neutrophil infiltrate in breast tumors. These data point to a mechanism by which NFAT1 orchestrates the communication between breast cancer cells and host neutrophils during breast cancer progression.

The microtubule inhibiting agent epothilone B antagonizes glioma cell motility associated with reorganization of the actin-binding protein α-actinin 4.

Invasion of normal brain tissue by brain tumor cells is a major contributing factor to the recurrence and resistance of clinically diagnosed glioblastomas to therapy (surgery, chemotherapy, radiation). Here, we have assessed the efficacy of the microtubule inhibiting agent epothilone B on glioblastoma cell motility, a prerequisite cellular program of invasive glioblastomas. Using cell migration assays and immunofluorescence techniques we demonstrated that epothilone B abrogated glioblastoma cell motility as a consequence of α-actinin 4 redistristrubiton and the breakdown of cellular structures (leading edge, stress fibers) it is associated with during cell migration. Evaluation of the microtubule actin cross linking factor in glioblastoma cells also revealed epothilone B invoked changes in this cytoskeleton cross linking protein, resembling α-actinin 4 changes in response to epothilone B. We have demonstrated in this study that epothilone B antagonizes glioblastoma cell motility due to the disruption of cytoskeleton binding proteins that aide in preserving the structural organization of the cytoskeleton filamentous network. Furthermore, we provide preclincial evidence that epothilone B effects on glioblastomas are not limited to the impairment of dividing tumors cells but that it also targets migratory and invasive glioblastoma cells, suggesting that this agent has potential clinical benefit due to its ability to target divergent cellular programs in the glioblastoma tumor mass.