Anchorage-independent Survival Research Articles

Abstract 6995: Functional characterization of the novel cell adhesion molecule MPZL3 in ovarian cancer

Abstract Ovarian cancer (OVCA) is the most lethal gynecological malignancy in the United States often detected at advanced stages. OVCA progression involves transcoelomic metastasis, where cells disseminate into the peritoneal fluid, adhere to form multicellular aggregates that promote anchorage-independent survival and facilitate metastatic colonization of the peritoneum. To meet the requirements of each stage of this detachment, aggregation, and re-attachment cycle, OVCA cells have been shown to dynamically regulate the expression of multiple cell adhesion molecules (CAMs) throughout their progression. Moreover, targeting cell-cell adhesion molecules has shown to be an effective method to slow/inhibit the progression of OVCA. In this study, we characterize the novel role of the understudied CAM MPZL3 in OVCA. Myelin protein zero-like 3 (MPZL3) is a transmembrane protein with homology to other immunoglobulin-like (Ig) family of CAMs. While it has been reported that altered Ig-CAM expression plays a role in ovarian cancer, the function of MPZL3 has not been investigated. Interestingly, TCGA data demonstrates that chromosomal loss of the MPZL3 locus (11q23.3) is frequently detected in several cancer types, including 22% of clear cell and 30% of high grade serous ovarian cancers (HGSOC), implying that loss of genes located in this area has tumorigenic consequences. On the other hand, few studies showed that MPZL3 contains an oncogenic potential in breast and MET-amplified cancer cell lines, suggesting that the role of MPZL3 in cancer is context dependent. To study the function of MPZL3 in OVCA, we used shRNA mediated MPZL3 knockdown in OVCAR4 human HGSOC cells and examined transcriptome-wide effects by RNA-sequencing. The subsequent Gene Set Enrichment Analysis indicated that pathways involved in regulating cell growth, death, adhesion, and epithelial-mesenchymal transition were significantly altered by loss of MPZL3. To validate the candidate pathways, we knocked down or over-expressed MPZL3 in OVCA cells to test its importance in regulating OVCA cell growth, apoptosis, adhesion, and migration. We found that loss of MPZL3 resulted in decreased cell growth with a concomitant resistance to both Cisplatin and Olaparib treatments, both of which are commonly used for treating OVCA. Moreover, we show that knockdown of MPZL3 alters the homotypic adhesive-capacity of OVCA cells. Ongoing studies are underway to further characterize the role played by MPLZ3 in the mentioned contexts. Additionally, to elucidate the underlying molecular mechanism and identify novel interactors of MPZL3, we will conduct co-immunoprecipitation experiments in OVCA cells expressing V5-tagged MPZL3. Understanding the novel role of MPZL3 will provide further insight into OVCA progression, thus generating opportunities of developing new treatments for patients with low MPZL3 expression as an approach for precision cancer medicine. Citation Format: Ya-Yun Cheng, Beth Worley, Zaineb Javed, Amal T. Elhaw, Priscilla W. Tang, Nadine Hempel. Functional characterization of the novel cell adhesion molecule MPZL3 in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6995.

Abstract A028: Investigating the role of SOX2 in promoting anchorage-independent survival of ovarian cancer cells

Abstract Ovarian cancer (OVCA) is the most lethal gynecologic malignancy, often detected at an advanced stage. OVCA progression involves transcoelomic metastasis, where tumor cells disseminate into the peritoneal ascites. Malignant cells in the ascites adapt to survive under Anchorage-Independent (A-I) conditions by altering the expression of genes escalating anoikis (A-I cell death) resistance and metastasis. Our lab has shown that OVCA cells manipulate their transcriptomic profile to promote the expression of anoikis resistance genes in A-I. Expression profiling of genes upregulated in A-I revealed the transcription factor SRY-Box Transcription Factor 2 (SOX2) to be highly upregulated in response to OVCA cell detachment. While we have previously demonstrated that SOX2 is necessary for the A-I survival of OVCA cells, the specific mechanism underlying SOX2-dependent A-I survival remains unknown. RNA-sequencing studies performed in the high-grade serous ovarian cancer cell line, OVCAR3, revealed that the Leucine-rich repeat-containing G-protein coupled receptor 5, LGR5, is the most significantly upregulated gene in A-I compared to attached conditions. Importantly, amongst those genes that are upregulated in A-I, LGR5 is significantly downregulated following SOX2 knockdown in A-I. LGR5 is an ‘orphan’ G-protein coupled receptor known to modulate canonical Wnt signaling upon interacting with its ligand R-spondin1 (RSPO1). Time-course experiments in OVCAR3 cells revealed a striking increase in LGR5 expression that peaked after 24 hours in A-I. Furthermore, siRNA mediated knockdown of SOX2 downregulated LGR5 expression only in A-I, suggesting that LGR5 is a potential SOX2 target under A-I conditions. Interestingly, bioinformatics analysis of the existing OVCA datasets from the Cancer Genome Atlas showed increased expression of RSPO1, which correlated with a significant decrease in the overall survival of OVCA patients. Interaction of LGR5 with its ligand RSPO1 is crucial for the receptor to signal through the Wnt pathway to promote cell survival and cancer stemness. Although SOX2 and LGR5 are established markers of cancer stemness in various tumor types, their direct relationship in promoting tumor progression and regulation of OVCA A-I cell survival remains unknown. Our data suggests that increased SOX2 and LGR5 expression could drive OVCA cell survival under A-I conditions. Our current studies focus on understanding the molecular mechanisms behind SOX2-driven regulation of LGR5 and the role of LGR5 in driving OVCA A-I cell survival. Citation Format: Shriya Kamlapurkar, Coryn Stump, Mythreye Karthikeyan, Nadine Hempel. Investigating the role of SOX2 in promoting anchorage-independent survival of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A028.

Abstract A018: Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival and metastasis of HER2-positive and basal breast cancer through regulation of caspase 14

Abstract Despite improvements in therapies, metastasis still leads to high mortality rates in breast cancer (BC). Consequently, discovering new therapies that are effective at targeting metastatic disease remains a critical need, particularly for the aggressive HER2+ and basal BC subtypes. The ability of cancer cells to survive in the absence of an extracellular matrix – termed anchorage-independent survival (AIS) – is central to the metastatic cascade and has emerged as a target biology of interest for reducing metastatic burden. Previously, we identified the SL enzyme dihydroceramide desaturase 1 (DES1) as a critical node for AIS of HER2+ BC cells. Here, we have extended these data to show that targeting DES1 through pharmacological inhibitors (4HPR), siRNA, or CRISPR-cas9 knockout was able to overcome AIS in basal BC cells as well as HER2+ BC cells that, functionally, led to a reduction of in vitro colony formation in soft agar. Extending these findings in vivo, we found that DES1 knockout led to a significant reduction in lung metastasis of both HER2+ and basal BC cells in murine models of experimental metastasis. Moreover, treatment of PyMT mice with the DES1 inhibitor 4HPR was able to reduce metastatic burden. To define downstream effectors of DES1 required for AIS, we performed a microarray analysis of control and DES1 knockout cells in suspension, leading us to identify caspase 14 as a downstream effector of DES1 in HER2+ and basal BC. Consistent with these findings, siRNA knockdown of caspase 14 led to a striking decrease in cell viability in colony formation assays. Finally, analysis of publicly available data found that high levels of DES1 and Caspase 14 were associated with poor clinical outcomes in both HER2+ and basal BC. Taken together, these data establish DES1 as a mediator of BC metastasis, likely through promoting AIS, and define caspase 14 as a target of DES1-driven signaling. Furthermore, results suggest that both DES1 and caspase 14 expression may be useful as biomarkers of metastasis-prone tumors. Citation Format: Deanna M Peperno, Danielle Lambadis, Victoria Alvarado, Andrew E Resnick, Christopher J Clarke. Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival and metastasis of HER2-positive and basal breast cancer through regulation of caspase 14 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A018.

Dielectrophoretic enrichment of live chemo-resistant circulating-like pancreatic cancer cells from media of drug-treated adherent cultures of solid tumors.

Due to low numbers of circulating tumor cells (CTCs) in liquid biopsies, there is much interest in enrichment of alternative circulating-like mesenchymal cancer cell subpopulations from in vitro tumor cultures for utilization within molecular profiling and drug screening. Viable cancer cells that are released into the media of drug-treated adherent cancer cell cultures exhibit anoikis resistance or anchorage-independent survival away from their extracellular matrix with nutrient sources and waste sinks, which serves as a pre-requisite for metastasis. The enrichment of these cell subpopulations from tumor cultures can potentially serve as an in vitro source of circulating-like cancer cells with greater potential for scale-up in comparison with CTCs. However, these live circulating-like cancer cell subpopulations exhibit size overlaps with necrotic and apoptotic cells in the culture media, which makes it challenging to selectively enrich them, while maintaining them in their suspended state. We present optimization of a flowthrough high frequency (1 MHz) positive dielectrophoresis (pDEP) device with sequential 3D field non-uniformities that enables enrichment of the live chemo-resistant circulating cancer cell subpopulation from an in vitro culture of metastatic patient-derived pancreatic tumor cells. Central to this strategy is the utilization of single-cell impedance cytometry with gates set by supervised machine learning, to optimize the frequency for pDEP, so that live circulating cells are selected based on multiple biophysical metrics, including membrane physiology, cytoplasmic conductivity and cell size, which is not possible using deterministic lateral displacement that is solely based on cell size. Using typical drug-treated samples with low levels of live circulating cells (<3%), we present pDEP enrichment of the target subpopulation to ∼44% levels within 20 minutes, while rejecting >90% of dead cells. This strategy of utilizing single-cell impedance cytometry to guide the optimization of dielectrophoresis has implications for other complex biological samples.

MUC13 drives cancer aggressiveness and metastasis through the YAP1-dependent pathway.

Anchorage-independent survival after intravasation of cancer cells from the primary tumor site represents a critical step in metastasis. Here, we reveal new insights into how MUC13-mediated anoikis resistance, coupled with survival of colorectal tumor cells, leads to distant metastasis. We found that MUC13 targets a potent transcriptional coactivator, YAP1, and drives its nuclear translocation via forming a novel survival complex, which in turn augments the levels of pro-survival and metastasis-associated genes. High expression of MUC13 is correlated well with extensive macrometastasis of colon cancer cells with elevated nuclear YAP1 in physiologically relevant whole animal model systems. Interestingly, a positive correlation of MUC13 and YAP1 expression was observed in human colorectal cancer tissues. In brief, the results presented here broaden the significance of MCU13 in cancer metastasis via targeting YAP1 for the first time and provide new avenues for developing novel strategies for targeting cancer metastasis.

MET exon 14 skipping mutation is a hepatocyte growth factor (HGF)-dependent oncogenic driver in vitro and in humanized HGF knock-in mice.

Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in non-transformed human lung cells, and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanized HGF knock-in strain of mice and further detected in tumor cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.

ALDOC- and ENO2- driven glucose metabolism sustains 3D tumor spheroids growth regardless of nutrient environmental conditions: a multi-omics analysis

BackgroundMetastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions.Methods3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2 to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines.ResultsWe found that the transition from 2 to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production, viability and size of 3D tumor spheroids produced by H460, HCC827, MCF7, and T47D cell lines.ConclusionsOur results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid for all lung and breast cancer cell lines we have analyzed in different nutrient environmental conditions. broader Validation of this mechanism in other cancer cells of different origin will be necessary to broaden the role of ALDOC and ENO2 to other tumor types. Future in vivo studies will be necessary to assess the role of ALDOC and ENO2 in cancer metastasis.

Abstract A034: Orchestrated expression of the atypical Rho-GTPase, RHOV, in response to matrix detachment of ovarian cancer cells

Abstract Ovarian cancer (OVCA) metastasis occurs through the process of transcoelomic spread, where cells disseminate into the peritoneal fluid and form multi-cellular aggregates (MCA) that mediate Anchorage-Independent (A-I) survival and invasion of the peritoneal organs. Isolation of MCAs from patient ascites has been extensively reported in the literature and found to correlate with poor patient survival and resistance to therapy. However, the mechanisms promoting OVCA MCA formation and survival in A-I are not fully understood. We previously demonstrated that MCAs upregulate their mitochondrial antioxidant defenses to maintain optimal survival in A-I, and that this is an early event following cellular detachment. To identify additional key molecular players promoting MCA formation and fitness in A-I, we employed sequential RNA seq analysis to compare the transcriptome of cells in attached, early A-I, and late A-I conditions using the serous cell line OVCA433. Analysis of early transcriptomic changes observed within 2hrs of A-I revealed that RHOV, a recently identified atypical member of the Rho GTPase family, is the top gene significantly upregulated in early A-I. Moreover, we found that RHOV expression rapidly declines in later A-I timepoints indicative of a tight temporal regulation of RHOV expression following cellular detachment. Next, we compared the expression of RHOV in primary ovarian tumor cells and matching malignant ascites cells from the publicly available dataset (GEO: GSE85296) and found increased RHOV expression specifically in the metastatic ascites of ovarian cancer patients when compared to the attached tumor. Previously, RHOV expression was found to regulate intercellular adhesion dynamics during specific stages of neural crest development, a physiological process mimicking epithelial to mesenchymal transition (EMT). RHOV has also been reported to be overexpressed in lung cancer where it was found to correlate with poor patient outcome and resistance to therapy. However, the role of RHOV in ovarian cancer metastasis remains unexplored. To test the consequences of increased RHOV transcription in A-I: its role in migration, aggregate formation, and anoikis resistance is being tested following silencing of RHOV expression using both CRISPR and siRNA. Current work is exploring how RHOV is rapidly transcribed following matrix detachment and how increased expression alters cellular signaling events in early A-I stages to promote optimal MCA fitness. This work aims to contribute new knowledge on the novel oncogenic role of an understudied member of the Rho family of GTPases, RHOV. Moreover, given that increased MCA fitness has been correlated with increases metastatic potential and resistance to therapy, we provide a unique proof-of-concept study to target key components of early MCA adaptations as a novel therapeutic strategy for prolonging survival rates in OVCA patients diagnosed in metastatic stages. Citation Format: Amal T. Elhaw, Yeon Soo Kim, Zaineb Javed, Priscilla Tang, Weihua Pan, Nadine Hempel. Orchestrated expression of the atypical Rho-GTPase, RHOV, in response to matrix detachment of ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A034.

Polyploid/Multinucleated Giant and Slow-Cycling Cancer Cell Enrichment in Response to X-ray Irradiation of Human Glioblastoma Multiforme Cells Differing in Radioresistance and TP53/PTEN Status.

Radioresistance compromises the efficacy of radiotherapy for glioblastoma multiforme (GBM), the most devastating and common brain tumor. The present study investigated the relationship between radiation tolerance and formation of polyploid/multinucleated giant (PGCC/MGCC) and quiescent/senescent slow-cycling cancer cells in human U-87, LN-229, and U-251 cell lines differing in TP53/PTEN status and radioresistance. We found significant enrichment in MGCC populations of U-87 and LN-229 cell lines, and generation of numerous small mononuclear (called Raju cells, or RJ cells) U-87-derived cells that eventually form cell colonies, in a process termed neosis, in response to X-ray irradiation (IR) at single acute therapeutic doses of 2-6 Gy. For the first time, single-cell high-content imaging and analysis of Ki-67- and EdU-coupled fluorescence demonstrated that the IR exposure dose-dependently augments two distinct GBM cell populations. Bifurcation of Ki-67 staining suggests fast-cycling and slow-cycling populations with a normal-sized nuclear area, and with an enlarged nuclear area, including one resembling the size of PGCC/MGCCs, that likely underlie the highest radioresistance and propensity for repopulation of U-87 cells. Proliferative activity and anchorage-independent survival of GBM cell lines seem to be related to neosis, low level of apoptosis, fraction of prematurely stress-induced senescent MGCCs, and the expression of p63 and p73, members of p53 family transcription factors, but not to the mutant p53. Collectively, our data support the importance of the TP53wt/PTENmut genotype for the maintenance of cycling radioresistant U-87 cells to produce a significant amount of senescent MGCCs as an IR stress-induced adaptation response to therapeutic irradiation doses.

SIRT3 supports anchorage-independent survival of ovarian cancer cells by regulating mitochondrial redox signaling

SIRT3 supports anchorage-independent survival of ovarian cancer cells by regulating mitochondrial redox signaling

Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival downstream of HER2-driven glucose uptake and metabolism.

Oncogenic reprogramming of cellular metabolism is a hallmark of many cancers, but our mechanistic understanding of how such dysregulation is linked to tumor behavior remains poor. In this study, we have identified dihydroceramide desaturase (DES1)-which catalyzes the last step in de novo sphingolipid synthesis-as necessary for the acquisition of anchorage-independent survival (AIS), a key cancer enabling biology, and establish DES1 as a downstream effector of HER2-driven glucose uptake and metabolism. We further show that DES1 is sufficient to drive AIS and in vitro tumorigenicity and that increased DES1 levels-found in a third of HER2+ breast cancers-are associated with worse survival outcomes. Taken together, our findings reveal a novel pro-tumor role for DES1 as a transducer of HER2-driven glucose metabolic signals and provide evidence that targeting DES1 is an effective approach for overcoming AIS. Results further suggest that DES1 may have utility as a biomarker of aggressive and metastasis-prone HER2+ breast cancer.

Understanding the cell survival mechanism of anoikis-resistant cancer cells during different steps of metastasis.

Anchorage-independent survival of cancer cells is associated with metastasis as it enables cells to travel to secondary target sites. Tissue integrity is generally maintained by detachment-induced cell death called 'anoikis', but cancer cells undergoing the multistep metastatic process show resistance to anoikis. Anoikis resistance enables these cells to survive through the extracellular matrix (ECM) deprived phase, which starts when cancer cells detach and move into the circulation till cells reach to the secondary target site. Comprehensive analysis of the molecular and functional biology of anoikis resistance in cancer cells will provide crucial details about cancer metastasis, enabling us to identify novel therapeutic targets against cancer cell dissemination and ultimately secondary tumor formation. This review broadly summarizes recent advances in the understanding of cellular and molecular events leading to anoikis and anoikis resistance. It further elaborates more about the signaling cross-talk in anoikis resistance and its regulation during metastasis.

Reciprocal SOX2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer.

Growth factors in tumor environments are regulators of cell survival and metastasis. Here, we reveal the dichotomy between TGF-β superfamily growth factors BMP and TGF-β/activin and their downstream SMAD effectors. Gene expression profiling uncovers SOX2 as a key contextual signaling node regulated in an opposing manner by BMP2, -4, and -9 and TGF-β and activin A to impact anchorage-independent cell survival. We find that SOX2 is repressed by BMPs, leading to a reduction in intraperitoneal tumor burden and improved survival of tumor-bearing mice. Repression of SOX2 is driven by SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2's promoter. Conversely, TGF-β, which is elevated in patient ascites, and activin A can promote SOX2 expression and anchorage-independent survival by SMAD3-dependent histone H3K4me3 recruitment. Our findings identify SOX2 as a contextual and contrastingly regulated node downstream of TGF-β members controlling anchorage-independent survival and metastasis in ovarian cancers.

Abstract 128: SIRT3 supports anchorage-independent survival of ovarian cancer cells

Abstract Metabolic changes during tumor progression have numerous consequences on cancer cells, including an increased risk of cell death due to production of reactive oxygen species (ROS). Antioxidant defense adaptations thus accompany metabolic alterations as essential survival mechanisms during metastasis. We observe that oxidative phosphorylation is increased when ovarian cancer (OC) cells transition to anchorage independence (a-i), an important step during metastasis in the peritoneal cavity. To combat the rise in oxidative stress resulting from this metabolic shift, a rapid increase in the sirtuin 3 (SIRT3)/superoxide dismutase 2 (SOD2) axis occurs to reduce the mitochondrial superoxide anion surge and to maintain cellular viability. We demonstrate that this upregulation occurs in transcription, translation, and SIRT3-mediated regulation of SOD2 activity. However, the fate of the hydrogen peroxide (H2O2) resulting from superoxide dismutation through SIRT3/SOD2 remains unknown. RNAseq analysis of attached versus a-i conditions revealed a significant downregulation of peroxiredoxin 3, a major scavenger of H2O2 in the mitochondria, suggesting that OCs have a net increase in mitochondrial H2O2 in a-i. At sub-lethal levels, H2O2 serves as a second messenger of redox signaling, primarily via protein cysteine oxidation, which has been implicated with metastasis. We thus hypothesize that increases in SIRT3/SOD2/H2O2 play a role as mediators of redox signaling during OC anchorage-independent survival, spheroid formation and metastasis. Using the HyPer7 redox probe and manipulation of SIRT3 and SOD2 expression, H2O2 generation and localization in a-i are being assessed using 3D fluorescence microscopy. Moreover, siRNA mediated knock-down of SIRT3 in a-i leads to the downregulation of genes involved in specific signaling pathways, including NF-kB signaling, the inflammatory response, and metabolism. Mass-spec studies are underway to identify specific SIRT3 target proteins upstream of these transcriptional changes, by assessing global cysteine oxidation and the cellular acetylome to determine the role of H2O2-dependent redox signaling and SIRT3-dependent protein de-acetylation, respectively. Given the increase in SIRT3-dependent inflammatory signaling in a-i, we are exploring how the SIRT3/SOD2/H2O2 axis influences OC interaction with tumor associated macrophages and mesenchymal stem cells, commonly found in the ascites tumor environment. This work demonstrates a multi-faceted role for the SIRT3/SOD2/H2O2 axis in a-i survival, a key step during OC metastasis. Citation Format: Priscilla Tang, Yeon Soo Kim, Piyushi Gupta-Vallur, Amal T. Elhaw, Zaineb Javed, Weihua Pan, Nadine Hempel. SIRT3 supports anchorage-independent survival of ovarian cancer cells [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 128.

Abstract 992: MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. About 90% of all cancer-related deaths are due to the development of metastatic sites in the body. 40-50% of colorectal cancer patients develop metastasis at some point during their fight with the disease. Understanding the mechanism of metastasis in colorectal cancer is vital. Metastasis is a multistep process; anchorage-independent survival after intravasation of cells from the primary tumor site is a crucial step. In our lab, we have previously demonstrated that MUC13 plays an essential role in the CRC progression by modulating anti-apoptotic pathways and survival proteins expression. Under anchorage-independent growth conditions, we have identified that MUC13, in correspondence with YAP1, is the key upregulated protein and is responsible for increased survival. Isogenic CRC cell lines SW480 (non-metastatic) and SW620 (metastatic) on low adherence growth conditions are applied in these studies. In addition, lentiviral transduced stable overexpression and knockdown cell lines were generated for MUC13 and YAP1 mechanistic studies. The overexpression of MUC13 in non-metastatic SW480 cells (low MUC13 expressing) increased anchorage-independent survival and enhanced tumorigenesis compared to SW480+Vector cells, contrary results were found upon MUC13 knockdown in SW620 cells (high MUC13 expressing). In vitro results were recapitulated in the in vivo mouse model system and human CRC tissues. In Proximity Ligation Assays (PLA), we found an increased nuclear localization of the survival complex YAP1/β-catenin in MUC13 overexpressing cells. In contrast, MUC13 knockdown resulted to the lower aboudance of survival complex in the nucleus. Immunoprecipitation validates the protein-protein interaction between MUC13 and YAP1. YAP1 knockdown in MUC13 overexpressing cells showed a decrease in survival, indicating the necessary functional complex formation between MUC13 and YAP1. MUC13 and YAP1 expression in human CRC tissue were highest at Stage II. However, YAP1 expression increased when MUC13 was observed in the nucleus. This supports the notion that MUC13 is critical in enhancing CRC metastasis through molecular interaction with YAP1. For the first time, this study demonstrates complex formation between MUC13 and YAP1 and defines their role in CRC progression and metastasis. Citation Format: Kyle Doxtater, Manish K. Tripathi, Radhika Sekhri, Sudhir Kotnala, Bilal Hafeez, Sheema Khan, Nadeem Zafar, Murali Yallapu, Meena Jaggi, Subhash Chauhan. MUC13 enhances colorectal cancer metastasis through molecular interaction with YAP1 transcription factor [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 992.

Abstract 2445: Orchestrated expression of the atypical Rho-GTPase RHOV mediates multicellular aggregate formation in ovarian cancer

Abstract Transcoelomic spread is a major mode of ovarian cancer (OVCA) metastasis, where cells disseminate into the peritoneal ascites fluid, evade anoikis, and form multi-cellular aggregates (MCAs) that mediate anchorage-independent (a-i) survival and invasion of the peritoneal organs. Isolation of MCAs from patient ascites correlates with poor patient survival and resistance to therapy. However, the mechanisms promoting anoikis evasion and OVCA MCA formation in a-i are not fully understood. Our previous data from patient ascites-derived cells, cell lines, and in vivo studies, demonstrate that OVCA cells upregulate the nutrient/stress sensor SIRT3 in response to a-i. We further illustrated that SIRT3 is necessary for the activation of the primary mitochondrial dismutase, SOD2, which in turn promotes the mitochondrial antioxidant defense to maintain optimal survival in a-i. To identify additional key molecular players important for a-i survival downstream of SIRT3, we employed RNA sequencing analysis to compare the transcriptome of SIRT3 knockdown and wildtype cells in both attached and a-i conditions using the serous adenocarcinoma cell line OVCA433. Analysis of early transcriptomic changes, observed within 2 hrs as well as 24 hrs of a-i, revealed that RHOV, a recently identified atypical member of the Rho GTPase family, is the top gene significantly upregulated in a-i. SIRT3 knock-down significantly decreases RHOV expression in 24hr a-i conditions, implicating RHOV as a potential downstream target of SIRT3 signaling in a-i. RHOV was reported to be overexpressed in lung cancer and found to correlate with poor patient outcome and resistance to therapy. RHOV expression also regulates intercellular adhesion dynamics during development. Our data show that genetic silencing of RHOV expression interferes with the ability of OVCA cells to form MCAs in a-i, suggesting that increased expression of RHOV in a-i conditions acts as an adaptive survival advantage mediating cell-cell adhesion and MCA formation. Current work is exploring how RHOV regulates E-cadherin in this context, by focusing on p21-stimulated serine/threonine protein kinase signaling. In addition, the mechanisms of SIRT3 dependent regulation are being explored by determining how changes in mitochondrial protein deacetylation contribute to RHOV transcription. This work highlights an expanding role of SIRT3 in mediating a-i survival of OVCA and explores the novel role of an understudied member of the Rho family of GTPases, RHOV during OVCA metastasis. Finally, given that the formation of MCAs has been correlated with resistance to therapy and increased rates of recurrence, we aim to test how MCA formation and targeting of key players of cell-cell adhesion in a-i, such as a RHOV, can be exploited for novel therapeutic strategies to prolong survival of OVCA patients diagnosed with metastatic disease. Citation Format: Amal T. Elhaw, Yeon Soo Kim, Zaineb Javed, Priscilla Tang, Weihua Pan, Nadine Hempel. Orchestrated expression of the atypical Rho-GTPase RHOV mediates multicellular aggregate formation in ovarian cancer [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 2445.

Abstract 2392: Adapted tumor suspension cells rewire metabolic pathways for anchorage independent survival

Abstract Metastatic spread of cancer cells is the main cause of cancer-related death. As cancer cells adapt themselves in a suspended state in the blood stream before penetration and regrowth at distal tissues, understanding their survival strategy in an anchorage-independent condition is important to develop appropriate therapeutics. We have previously generated adapted suspension cells (ASCs) from parental adherent cancer cells to study the characteristics of circulating tumor cells. In this study, we explored metabolic rewiring in MDA-MB-468 ASCs to adapt to suspension growth conditions through extracellular flux analyses and various metabolic assays. We also determined the relationship between protein kinase B (AKT) activation and metabolic rewiring in ASCs using the AKT inhibitor, MK2206. ASCs reprogramed metabolism to enhance glycolysis and basal oxygen consumption rate. RNA-sequencing analysis revealed the upregulation in the genes related to glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The changes in the metabolic program led to a remarkable dependency of ASCs on carbohydrates as an energy source for proliferation as compared to parental adherent cells (ADs). AKT activation was observed in ASCs and those generated from pancreatic and other breast cancer cells, and AKT activation inhibition in ASCs decreased glycolysis and oxygen consumption. AKT activation is an important strategy for obtaining energy through the enhancement of glycolysis in ASCs. The regulation of AKT activity and/or glycolysis may provide a new therapeutic strategy to prevent the metastatic spread of cancer cells. Citation Format: Young Yang, Hyun Jeong Ju. Adapted tumor suspension cells rewire metabolic pathways for anchorage independent survival [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 2392.

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.

Adapted suspension tumor cells rewire metabolic pathways for anchorage-independent survival through AKT activation

Metastatic spread of cancer cells is the main cause of cancer-related death. As cancer cells adapt themselves in a suspended state in the blood stream before penetration and regrowth at distal tissues, understanding their survival strategy in an anchorage-independent condition is important to develop appropriate therapeutics. We have previously generated adapted suspension cells (ASCs) from parental adherent cancer cells to study the characteristics of circulating tumor cells. In this study, we explored metabolic rewiring in MDA-MB-468 ASCs to adapt to suspension growth conditions through extracellular flux analyses and various metabolic assays. We also determined the relationship between AKT activation and metabolic rewiring in ASCs using the AKT inhibitor, MK2206. ASCs reprogramed metabolism to enhance glycolysis and basal oxygen consumption rate. RNA-sequencing analysis revealed the upregulation in the genes related to glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The changes in the metabolic program led to a remarkable dependency of ASCs on carbohydrates as an energy source for proliferation as compared to parental adherent cells (ADs). AKT activation was observed in ASCs and those generated from pancreatic and other breast cancer cells, and AKT activation inhibition in ASCs decreased glycolysis and oxygen consumption. AKT activation is an important strategy for obtaining energy through the enhancement of glycolysis in ASCs. The regulation of AKT activity and/or glycolysis may provide a strong therapeutic strategy to prevent the metastatic spread of cancer cells.

MiR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFβR3

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm invivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.