Anchorage-independent Cells Research Articles

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin β1/FAK signaling pathway

BackgroundTemozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PurposeThis study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. MethodsThe differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin β1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. ResultsDEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin β1, FAK, β-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin β1, β-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin β1/FAK pathway. ConclusionWe first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin β1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

FASN deficiency induces a cytosol-to-mitochondria citrate flux to mitigate detachment-induced oxidative stress

Fatty acid synthase (FASN) maintains de novo lipogenesis (DNL) to support rapid growth in most proliferating cancer cells. Lipogenic acetyl-coenzyme A (CoA) is primarily produced from carbohydrates but can arise from glutamine-dependent reductive carboxylation. Here, we show that reductive carboxylation also occurs in the absence of DNL. In FASN-deficient cells, reductive carboxylation is mainly catalyzed by isocitrate dehydrogenase-1 (IDH1), but IDH1-generated cytosolic citrate is not utilized for supplying DNL. Metabolic flux analysis (MFA) shows that FASN deficiency induces a net cytosol-to-mitochondria citrate flux through mitochondrial citrate transport protein (CTP). Previously, a similar pathway has been shown to mitigate detachment-induced oxidative stress in anchorage-independent tumor spheroids. We further report that tumor spheroids show reduced FASN activity and that FASN-deficient cells acquire resistance to oxidative stress in a CTP- and IDH1-dependent manner. Collectively, these data indicate that by inducing a cytosol-to-mitochondria citrate flux, anchorage-independent malignant cells can gain redox capacity by trading off FASN-supported rapid growth.

Abstract 1291: Signaling pathways modulated by lncRNA UCA1 during anchorage-independent growth in colorectal cancer

Abstract Colorectal cancer (CRC) is the second deadliest cancer, and over 40-50% of patients develop metastasis during their fight against the disease. CRC survival rate drops from 90% to 14% when the condition is contained within the colon vs. when found at distant sites within the body. Metastasis is a multistep process and one of the critical steps for cancer cells to acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory and lymphatic systems to distant target organs. Thus, understanding the molecular players involved in the anoikis process and metastasis could be vital for improving the survival of CRC patients. The aberrant expression of a long noncoding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has been identified in CRC. However, its role in metastasis processes is not yet well defined. Our preliminary results in the anchorage-independent growth (anoikis model) demonstrate increased expression of lncRNA UCA1. Moreover, the overexpression of lncRNA UCA1 led to high expression of stemness markers SOX2 and Kaiso, along with increased survival of anchorage-independent cells, which indicates a potential mechanistic role of UCA1 in anoikis resistance. Thus, in this study, we propose elucidating the role(s) of UCA1 and its associated signaling pathways during anoikis resistance. We hypothesize that the overexpression of lncRNA UCA1 enhances CRC metastasis through anoikis resistance-associated signaling pathways. We will utilize Isogenic CRC cell lines SW480 (oncogenic) and SW620 (metastatic) to understand the mechanistic regulation of anoikis resistance. Lentiviral transduced stable overexpression (SW480+UCA1//GFP) and knockdown (SW620+CRISPRgUCA1) cell lines have been utilized for this study. After subjecting these cell lines (along with control) to anchorage-independent growth conditions, cell cycle, pro-survival, anti-apoptotic, stemness, and glucose metabolism factors will be analyzed through RT-PCR, Digital Droplet PCR (ddPCR), western blot, and FACS analyses. Utilizing the same model, we will examine lncRNA UCA1 linked anoikis resistance specific phosphorylation profiles of kinases, their protein substrates using the Proteome Profiler Phospho-Human Phospho-Kinase Array. Citation Format: Sophia Leslie, Kyle D. Doxtater, Samantha Lopez, Kristopher Ezell, Adithya Anilkumar, Amayrani Sanchez, Bilal Hafeez, Tamer Oraby, Meena Jaggi, Timothy Loy, Subhash Chauhan, Manish K. Tripathi. Signaling pathways modulated by lncRNA UCA1 during anchorage-independent growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1291.

Conoidin A Abrogates Growth of Anchorage-Dependent and Anchorage-Independent SKOV3 Ovarian Cancer Cells

Ovarian cancer is one of the leading causes of cancer death in the United States with only 49% of women surviving 5 years after initial diagnosis. Poor screening methods, chemoresistance, and its high metastatic capacity make this disease difficult to treat. Thus, improved treatment methodologies are necessary to improve survival rates and quality of life for ovarian cancer patients. Conoidin A is a water-soluble covalent inhibitor of peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2). While its effects have been primarily studied on parasites, recent studies also begin to elucidate the impacts of conoidin A in other human health conditions, including anticancer effects. However, the anticancer effects and potential utility of conoidin A as a therapeutic, specifically in ovarian cancer, has yet to be investigated. Here, we report that conoidin A eliminates growth of anchorage-dependent and anchorage-independent SKOV3 cells. Furthermore, analyses of clinical samples show increased expression of PRDX1 and PRDX2 in ovarian cancerous tissue, which are the targets of conoidin A. These data identify conoidin A as a potential therapeutic for early and late-stage ovarian cancer and warrants further investigation. (First Online: December 31, 2022)

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

The Role of SOD2 in Migration and Anchorage-Independent Growth of SKOV3 Ovarian Cancer Cells

The 5-year survival rates for ovarian cancer are 93%, 75%, and 30% for localized, regional, and distant tumors, respectively. These vast differences in survival rates underscore the need to identify novel therapeutic targets that are effective at different stages of tumor progression to better treat all patients diagnosed with this deadly disease. Manganese superoxide dismutase (SOD2) is a mitochondrial antioxidant enzyme responsible for eliminating superoxide and preventing oxidative damage to the mitochondria. Recent studies have implicated changes in SOD2 expression levels in multiple cancers, including breast, colorectal, prostate, and head and neck. While studies have begun to unravel the role of SOD2 in ovarian cancer, no one has looked at the specific role SOD2 plays in distinct stages of tumor progression of ovarian cancer. Here, we report SOD2 deficiency (accomplished through utilizing shRNA techniques) results in increased invasive and migratory ability of SKOV3 cells, a commonly used ovarian adenocarcinoma cell line. In contrast, SOD2-deficient SKOV3 cells display abrogated anchorage-independent growth in soft agar. These studies in SKOV3 cells indicate that SOD2 expression hampers invasion and migration critical for early tumor initiation but helps maintain anchorage-independent growth necessary for ovarian cancer metastases. In aggregate, SOD2 plays a context-dependent role in ovarian cancer progression and its utility as a therapeutic target for later-stage anchorage-independent ovarian cancer cells should be further explored. (First online: March 1, 2022)

Targeting Oxidative Phosphorylation-Proteasome Activity in Extracellular Detached Cells Promotes Anoikis and Inhibits Metastasis.

Metastasis arises owing to tumor cells’ capacity to evade pro-apoptotic signals. Anoikis—the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells’ ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.

Pleiotropic Mechanisms Drive Endocrine Resistance in the Three-Dimensional Bone Microenvironment.

Although hormonal therapy (HT) inhibits the growth of hormone receptor-positive (HR+) breast and prostate cancers, HT resistance frequently develops within the complex metastatic microenvironment of the host organ (often the bone), a setting poorly recapitulated in 2D culture systems. To address this limitation, we cultured HR+ breast cancer and prostate cancer spheroids and patient-derived organoids in 3D extracellular matrices (ECM) alone or together with bone marrow stromal cells (BMSC). In 3D monocultures, antiestrogens and antiandrogens induced anoikis by abrogating anchorage-independent growth of HR+ cancer cells but exhibited only modest effects against tumor cells residing in the ECM niche. In contrast, BMSC induced hormone-independent growth of breast cancer and prostate cancer spheroids and restored lumen filling in the presence of HR-targeting agents. Molecular and functional characterization of BMSC-induced hormone independence and HT resistance in anchorage-independent cells revealed distinct context-dependent mechanisms. Cocultures of ZR75-1 and LNCaP with BMSCs exhibited paracrine IL6-induced HT resistance via attenuation of HR protein expression, which was reversed by inhibition of IL6 or JAK signaling. Paracrine IL6/JAK/STAT3-mediated HT resistance was confirmed in patient-derived organoids cocultured with BMSCs. Distinctly, MCF7 and T47D spheroids retained ER protein expression in cocultures but acquired redundant compensatory signals enabling anchorage independence via ERK and PI3K bypass cascades activated in a non-IL6-dependent manner. Collectively, these data characterize the pleiotropic hormone-independent mechanisms underlying acquisition and restoration of anchorage-independent growth in HR+ tumors. Combined analysis of tumor and microenvironmental biomarkers in metastatic biopsies of HT-resistant patients can help refine treatment approaches. SIGNIFICANCE: This study uncovers a previously underappreciated dependency of tumor cells on HR signaling for anchorage-independent growth and highlights how the metastatic microenvironment restores this malignant property of cancer cells during hormone therapy.

A Positive Feedback Loop Between TGFβ and Androgen Receptor Supports Triple-negative Breast Cancer Anoikis Resistance.

Triple-negative breast cancer (TNBC) is an aggressive subtype with peak recurrence as metastatic disease within the first few years of diagnosis. Androgen receptor (AR) expression is increased in anchorage-independent cells in TNBC preclinical models. Both AR knockdown and inhibition lead to reduced TNBC invasion in vitro, reduced tumorigenicity, and less recurrence in vivo in preclinical models. Transforming growth factor β (TGFβ) pathway gene signatures also increased during anchorage-independent survival both in vitro and in vivo in preclinical models and in circulating tumor cells (CTCs) from patients during emergence of chemo resistant disease. We hypothesized that a positive loop between AR and TGFβ signaling facilitates TNBC anchorage-independent survival. We find that multiple components of the TGFβ pathway, including TGFβ1 and 3, as well as pathway activity measured by nuclear localization and transcriptional activity of phosphorylated Smad3, are enhanced in anchorage-independent conditions. Further, exogenous TGFβ increased AR protein while TGFβ inhibition decreased AR and TNBC viability, particularly under anchorage-independent culture conditions. ChIP-seq experiments revealed AR binding to TGFB1 and SMAD3 regulatory regions in MDA-MB-453 cells. In clinical datasets, TGFB3 and AR positively correlate and high expression of both genes together corresponded to significantly worse recurrence-free and overall survival in both ER-negative and basal-like breast cancer. Finally, inhibiting both AR and TGFβ decreased cell survival, particularly under anchorage-independent conditions. These findings warrant further investigations into whether combined inhibition of AR and TGFβ pathways might decrease metastatic recurrence rates and mortality from TNBC.

High-Throughput Measurements of Intra-Cellular and Secreted Cytokine from Single Spheroids Using Anchored Microfluidic Droplets.

While many single-cell approaches have been developed to measure secretions from anchorage-independent cells, these protocols cannot be applied to adherent cells, especially when these cells require to be cultured in 3D formats. Here, a platform to measure secretions from individual spheroids of human mesenchymal stem cells, cultured within microfluidic droplets is introduced. The platform allows to quantify the secretions from hundreds of individual spheroids in each device, by using a secondary droplet to bring functionalized micro-beads in proximity to each spheroid. Vascular endothelial growth factor (VEGF-A) is measured on and a broad distribution of secretion levels within the population of spheroids is observed. The intra-cellular level of VEGF-A on each spheroid, measured through immuno-staining, correlates well with the extra-cellular measurement, indicating that the heterogeneities observed at the spheroid level result from variations at the intra-cellular level. Further, the molecular accumulation within the droplets is modeled and it is found that physical confinement is crucial for measurements of protein secretions. The model predicts that the time to achieve a measurement scales with droplet volume. These first measurements of secretions from individual spheroids provide several new biological and technological insights.

Abstract 332: Nanofibrillar cellulose hydrogel as 3D culture matrix towards the development of tumor models

Abstract Introduction. Physiologically relevant and robust in vitro cell models are needed for cancer model development, as well as standard cell-based drug screening assays. These models and assays may require the use of well-established cancer cell-lines or primary patient-derived cells. It is well known that three-dimensional (3D) cell culture provides a more in vivo -like environment for cells compared to traditional 2D monolayer culture. GrowDex®, a nanofibrillar cellulose (NFC) hydrogel, derived from the Birch tree, has been shown to reproducibly support the culture of many different cell types in 3D. Many animal derived hydrogels contain growth factors, other biological signaling molecules and unknown amounts of different ECM proteins, and have also suffered from variation between lots. The tunable stiffness, shear thinning properties and room-temperature stability, enable GrowDex to be used in a multitude of applications with a wide variety of different cell types and tissues. Additionally, cellulase enzyme (GrowDase™) treatment allows the recovery of cells or multicellular structures without impact to cell viability, phenotype or function. Methods and Results. Cancer cells derived from either solid tumor (e.g. osteosarcoma or breast cancer) or liquid biopsies (e.g. pleural or ascites effusion) require different culturing conditions. Studies have shown that GrowDex provides a suitable inert matrix where anchorage dependent and independent cancer cells can be cultured for short- or long-term studies, without observing uncontrolled matrix induced effects. Cells were seen to form round well-defined spheroids or multicellular structures. Additionally, co-cultures of cancer cells with fibroblasts provided a more physiologically relevant environment for the investigation of cell-cell effects on microtissue formation. When cells were seeded with different cell ratios, microtissues showed different morphologies and sizes. GrowDex has also been used successfully for the culture and characterization of an intensively pigmented cell line derived from a highly aggressive NRAS mutated cutaneous melanoma. Cultured cells maintained their natural in vivo behavior and morphology, as well as increasing the deep brown pigmentation staining. In addition, GrowDex has been shown to be an optimum matrix for use with patient derived cancer (PDC) cells cultured for use in drug screening tests for personalized medicine. Differences in drug responses with the PDC cells in 2D and 3D conditions were observed. Conclusions. GrowDex is highly biocompatible with over150 cell types including human cancer cells and primary tumor tissue. The hydrogel is animal-free, ready-to-use with shear-thinning properties making it easy to handle and automate providing a well-defined 3D environment for drug screening and cell-based disease model development assays. Citation Format: Jonathan Sheard. Nanofibrillar cellulose hydrogel as 3D culture matrix towards the development of tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 332.

Cholesterol grafted cationic lipopolymers: Potential siRNA carriers for selective chronic myeloid leukemia therapy.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase SOD2. This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 up-regulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

Catalase Deficiency Compromises Survival in Extracellular Matrix-Detached SKOV3 Ovarian Cancer Cells

For epithelial ovarian cancer cells to survive during the metastatic cascade, cells must be able to evade anoikis, a caspase-dependent cell death mechanism initiated by extracellular matrix (ECM) detachment. However, many of the details behind this phenomenon have yet to be unveiled. Here, we examined the role of the antioxidant enzyme, catalase, in the survival and proliferation of anchorage-independent SKOV3 ovarian cancer cells. Catalase deficiency severely compromises cell viability and anchorage-independent growth in ECM-detached SKOV3 cells. Notably, cellviability and proliferation were unaffected in ECM-attached catalase-deficient SKOV3 cells. In aggregate, we discovered that catalase plays a prominent role in protection from ECM-detachment-induced cell death in SKOV3 cells. Furthermore, these findings imply that catalase may be an effective therapeutic target for epithelial ovarian cancer cells that survive the ECM-bereft metastatic cascade.

Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers.

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvβ3, galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvβ3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvβ3-expressing KRAS-mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancers, and indicates integrin αvβ3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvβ3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvβ3 to promote KRAS-mediated activation of AKT. Cancer Discov; 7(12); 1464-79. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

136 - Mitochondrial Function is Dynamically Regulated during Ovarian Cancer Progression through Alterations in Fusion/Fission

Ovarian cancer continues to be the most deadly gynecological malignancy. A large proportion of patients are diagnosed at late stage, when significant metastasis has occurred through the intraperitoneal (IP) cavity. Preliminary work suggests that changes in mitochondrial function play important roles in tumor growth, metastasis and chemoresistance, and that this can be dynamically regulated dependent on tumor stage. Our data suggest that under proliferative attached conditions the high-grade serous adenocarcinoma cell line OVCA420 can shut-down mitochondrial function by altering its fission/fusion dynamics. Significant hyperfusion is associated with increased reliance on glycolysis and specific expression patterns of Drp1 splice variants, the GTPase responsible for mitochondrial fission. Of clinical relevance, a subgroup of cancer cells derived from patients diagnosed with late stage disease show similar patterns of Drp1 expression and mitochondria dysfunction. Characterization of these Drp1 splice variants is underway to determine their role as dominant negative fission proteins in ovarian cancer. Interestingly, we find that expression of Drp1 variants is dynamically regulated during different stages of ovarian cancer metastasis. Expression of Drp1 variants with apparent dominant negative function are decreased when cells are cultured as cellular spheroid aggregates, which models anchorage-independent survival and cells detaching from the primary tumor into the IP cavity during transcoelomic spread. This is accompanied by restoration of mitochondrial fission/ fusion dynamics and increased auto/mitophagy, suggesting that cancer cells in anchorage-independent conditions alter their fission protein expression to maintain a healthy pool of mitochondria, while expression of dominant negative Drp1 is associated with a highly proliferative cellular phenotype. In conclusion, dynamic Drp1 expression switching between the dominant-negative and functional Drp1 fission proteins may be a novel way for cancer cells to regulate fission/fusion, mitochondrial quality control and metabolism.

Monitoring cell substrate interactions in exopolysaccharide-based films reinforced with chitin whiskers and starch nanoparticles used as cell substrates

ABSTRACTExopolysaccharides (EPS) extracted from Nostoc commune cyanobacteria were used to prepare low-adhesion cell substrates for tumor-selective cell growth. The substrates were reinforced with chitin whiskers and starch nanoparticles and characterized using Fourier transform infrared spectroscopy, X-ray diffraction, and tensile tests. Two anchorage independent cells, green fluorescent protein-modified HeLa and THP-1 cells were seeded on the samples to assess their interaction with the composites. The results confirmed the cell–surface interaction of EPS-based composites. The mechanical properties of these films can be tailored by controlling the type and concentration of the reinforcement, which can be used in the design of different types of biomaterials.

Abstract 5464: Catalase mediates the survival of anchorage-independent ovarian cancer cells

Abstract Among United States women, ovarian cancer is the fifth leading cause of cancer-related death and the deadliest of the female reproductive system cancers. This is due to a lack of early detection, in effect, leading to primary diagnosis in later, metastasized stages for which there are few effective treatment options. In order to metastasize, ovarian cancer cells must be able to survive detachment from the extracellular matrix (ECM). When a non-tumorigenic mammary epithelial cell detaches from the ECM, research has shown that levels of Reactive Oxygen Species (ROS) rise in the cell and inhibit cell metabolism resulting in decreased cell survival. Previous research has also shown that ECM-detached breast cancer cells are able to utilize antioxidant enzymes to neutralize ROS thereby rescuing ATP levels and promoting cell survival. This antioxidant activity has also been shown to bolster cancer cells’ survival in anchorage independence. Although studied in breast cancer cells, no work has been done in ovarian cancer, which exhibits a very unique mode of metastasis through the peritoneal fluid. To begin to understand the role of antioxidant enzymes in metastatic ovarian cancer cells, we have worked to characterize the role of catalase, a prominent antioxidant enzyme, in the survival of ECM-detached SKOV3 ovarian cancer cells. In order to do so, we utilized short hairpin RNA (shRNA) to engineer SKOV3 cells deficient in catalase. The catalase-deficient cells were then tested for colony formation in detachment and cell viability and proliferation in both attachment and detachment. The results show that elimination of catalase expression does not have a significant effect on proliferation in attachment or detachment, but significantly decreases both cell viability and colony formation of SKOV3 cells grown in ECM-detachment. This suggests that elimination of catalase expression may hold promise for targeted treatment in late stage ovarian cancer, calling for further investigation of the mechanisms behind these results and the role that other antioxidant enzymes play in ovarian cancer. Future studies include looking at this phenomenon in other ovarian cancer cell lines, and using mouse xenograft studies to examine the effects of antioxidant compounds on survival of metastatic ovarian cancer cells. Citation Format: Cassandra L. Libbing, Calli A. Versagli. Catalase mediates the survival of anchorage-independent ovarian 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 5464. doi:10.1158/1538-7445.AM2017-5464

Abstract BS3-2: The unfolded protein response: A protective pathway and therapeutic target in breast cancer

Abstract We identified BHPI, a potent first-in-class non-competitive estrogen receptor α (ERα) biomodulator that kills therapy-resistant ER positive cancer cells. In mouse xenografts, BHPI induced rapid and dramatic regression of breast cancers. We also demonstrated BHPI's effectiveness in cancer cells containing ER mutations Y537S and D538G that are common in metastatic breast cancer. We used the CRISPR-Cas9 genome editing system to isolate T47D breast cancer cell lines in which both copies of wild type ER are replaced with ERY537S or ERD538G. In anchorage independent 3-dimensional culture, the ERY537S and ERD538G cells exhibit robust proliferation in micromolar 4-hydroxytamoxifen and fulvestrant/Faslodex/ICI 182,780, but are killed by nanomolar BHPI. BHPI is so effective because it works by distorting a newly identified normal pathway of estrogen-ER action. BHPI hyperactivates the endoplasmic reticulum stress sensor, the unfolded protein response (UPR). Estrogen-ER rapidly elicits moderate and transient anticipatory activation of the UPR. Activation of this conserved pathway is critical for estrogen and epidermal growth factor (EGF) induced gene expression and cell proliferation and for VEGF regulated angiogenesis and cell survival. Moreover, in analysis of data from approximately 1,000 ER positive breast cancer patients, activation of a UPR gene signature at diagnosis was tightly correlated with subsequent tumor recurrence, tamoxifen resistance and a poor prognosis. A major mechanism by which tumors develop resistance to therapy is overexpression of pumps, such as multidrug resistance protein 1 (MDR1)/p-glycoprotein, that carry out ATP-dependent pumping of anticancer drugs out of tumor cells. Nontoxic small molecule inhibitors targeting MDR1 remain elusive. Instead, we used BHPI's novel action in cancer cells to target MDR1's substrate, ATP. Acting via ER, BHPI hyperactivates the anticipatory UPR pathway leading to an ATP-depleting futile cycle in which there is ATP-dependent pumping of calcium into the endoplasmic reticulum (EnR), but the EnR calcium channels opened by BHPI-ER action allow the calcium to leak back out. This futile cycle rapidly depletes intracellular ATP, inactivating MDR1 in multidrug resistant breast and ovarian cancer cells. BHPI restored sensitivity of multidrug resistant breast and ovarian cancer cells to killing by therapeutically relevant concentrations of several anticancer drugs. In an orthotopic ovarian cancer xenograft model using OVCAR-3 cells, that are highly resistant to diverse anticancer drugs, the taxane paclitaxel was ineffective and the paclitaxel-treated tumors were uniquely prone to metastasize to adjacent tissue. BHPI alone strongly reduced tumor growth. Notably, tumors were undetectable in mice treated with BHPI plus paclitaxel. Compared to control ovarian tumors, after the combination therapy, plasma levels of the cancer biomarker, CA125, were at least several hundred fold lower; moreover, CA125 levels progressively declined to undetectable. Targeting drug resistance in ER positive tumors through BHPI-mediated, UPR-dependent, ATP depletion represents a promising new therapeutic strategy. (Support: NIH RO1 DK 071909, DOD BCRP W81XWH-13) Citation Format: Shapiro DJ. The unfolded protein response: A protective pathway and therapeutic target in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr BS3-2.

Biomaterials for polynucleotide delivery to anchorage-independent cells.

Anchorage-independent cells possess morphological features and cell membrane compositions that are distinct from adherent cells. They display minimal surface area, have a low rate of endocytosis and generally possess few proteoglycans which make it a challenge to deliver nucleic acids into them. Wide ranges of methods and materials have been developed to tackle the delivery obstacles for the polynucleotide-based therapeutics in modifying non-adherent cells. This article summarizes the techniques and biomaterials that have been utilized for transfection of anchorage-independent cells. First, physical techniques are briefly described along with particular applications for which they are well-suited. The structure-activity relationship of various biomaterial carriers of polynucleotides are then discussed with strategies employed to enhance their capability to transfect anchorage-independent cells. In conclusion, the authors' perspectives on different methods for polynucleotide delivery to primary human cells are compared, along with a discussion of their progression towards clinical trials.