Anchorage-independent Conditions Research Articles

Characterization of BTX-10908, a first-in-class, orally bioavailable SOS1 bifunctional degrader for the treatment of KRAS- and RTK-driven cancers.

e15192 Background: Despite early clinical responses of targeted therapeutics against specific nodes in the RTK-RAS-MAPK pathway, limitations in the depth and duration of efficacy occur due to the emergence of adaptive and acquired resistance mechanisms. Resistance can result from the removal of feedback inhibition to reactivate RTKs, on-target amplification and new mutations. The efficacy of these oncogene-targeted agents in the clinic could be improved by co-treatment with additional therapies that address either type of resistance mechanism. The RAS guanine nucleotide exchange factor, Son of Sevenless 1 (SOS1), represents an ideal target to enhance clinical potency of other pathway inhibitors for two reasons. In addition to mitigating inputs from upstream RTKs to downstream RAS proteins, SOS1’s activity is also regulated by ERK- and RSK-mediated negative feedback inhibition with upstream and downstream inhibitors alleviating this inhibition to reengage pathway activation. Methods: To address this need, we utilized our PRODEGY platform to generate orally bioavailable, Cereblon (CRBN)-based SOS1 bifunctional degrader, BTX-10908, for the treatment of KRAS- and RTK-driven tumors. Results: BTX-10908 demonstrated rapid and potent SOS1 degradation with <10nM DC50 and maximum degradation > 90%. Consistent with BTX-10908’s mechanism of action, SOS1 degradation required CRBN and the proteasome. Not only did it reduce active RAS levels, but BTX-10908 also inhibited downstream signaling proteins (pERK, pRSK, pS6) and lowered ERK-mediated transcripts in KRAS- and EGFR-mutant as well as MET amplified cell lines under anchorage-independent (3D) conditions. Additionally, drug washout experiments demonstrated prolonged suppression of pERK and ERK-regulated transcripts with SOS1 degradation whereas rebound occurred quicker with SOS1 inhibitors. BTX-10908 displayed CRBN- and SOS1-dependent antiproliferative activity with IC50 values ranging from 0.5-20nM. Notably, BTX-10908 was significantly more potent than the clinical SOS1 inhibitor at reducing pERK levels and cell viability in multiple KRAS- and RTK-driven cell lines. Oral administration of BTX-10908 resulted in dose-dependent SOS1 degradation and tumor growth inhibition in H358 (KRAS G12C) and H441 (KRAS G12V) xenografts. The combination of BTX-10908 with KRAS G12C inhibitors (Sotorasib, Adagrasib) and various RTK inhibitors (EGFR, Met, BCR-ABL, and FLT3) prevented pathway reactivation driven by these inhibitors resulting in synergistic effects in in vitro proliferation assays and enhanced tumor growth inhibition in KRAS-mutant xenograft models. Conclusions: Our developmental candidate, BTX-10908, displayed robust in vitro and in vivo activity supporting its further development for monotherapy and combination therapies of KRAS and RTK-driven cancers.

Delineating three-dimensional behavior of uveal melanoma cells under anchorage independent or dependent conditions

BackgroundAlthough rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention.MethodsSix UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis.ResultsUM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions.ConclusionsUnder an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

Epithelial-mesenchymal interaction protects normal colonocytes from 4-HNE-induced phenotypic transformation.

Recent studies have shown that epithelial-stromal interactions could play a role in the development of colorectal cancer. Here, we investigated the role of fibroblasts in the transformation of normal colonocytes induced by 4-HNE. Normal Co colonocytes and nF fibroblasts from the same mouse colon were exposed, in monoculture (m) or coculture (c), to 4-HNE (5 μM) twice weekly for 3 weeks. Gene expression was then analysed and the ability of Co colonocytes to grow in anchorage-independent conditions was tested in soft agar. Fibroblasts previously treated or not with 4-HNE were also seeded in culture inserts positioned above the agar layers to allow paracrine exchanges with colonocytes. First, 60% of the genes studied were modulated by coculture in Co colonocytes, with notably increased expression of BMP receptors. Furthermore, while 4-HNE increased the ability of monoculture-treated Co colonocytes to form colonies, this effect was not observed in coculture-treated Co colonocytes. Adding a selective BMPR1 inhibitor during the treatment phase abolished the protective effect of coculture. Conversely, addition of a BMP4 agonist to the medium of monoculture-treated Co colonocytes prevented phenotypic transformation by 4-HNE. Second, the presence of nF(m)-HNE fibroblasts during the soft agar assay increased the number and size of Co(m) colonocyte colonies, regardless of whether these cells had been previously treated with 4-HNE in monoculture. For soft agar assays performed with nF(c) and Co(c) cells initially treated in coculture, only the reassociation between Co(c)-HNE and nF(c)-HNE resulted in a small increase in the number of colonies. During the exposure phase, the epithelial-mesenchymal interaction protected colonocytes from 4-HNE-induced phenotypic transformation via activation of the BMP pathway. This intercellular dialogue also limited the ability of fibroblasts to subsequently promote colonocyte-anchorage-independent growth. In contrast, fibroblasts pre-exposed to 4-HNE in monoculture strongly increased the ability of Co(m) colonocytes to form colonies.

BRAFV600E promotes anchorage-independent growth but inhibits anchorage-dependent growth in hTERT/Cdk4-Immortalized normal human bronchial epithelial cells

Certain oncogenes, including mutant RAS and BRAF, induce a type of senescence known as oncogene-induced senescence (OIS) in normal cells in a cell-type-specific manner. OIS serves as a barrier to transformation by activated oncogenes. Our previous studies showed that mutant KRASV12 did not efficiently induce OIS in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line (HBEC3), but it did enhance both anchorage-dependent and anchorage-independent growth. In this study, we investigated whether mutant BRAF, a well-known inducer of OIS, could trigger OIS in HBEC3 cells. We also assessed the impact of mutant BRAF on the growth of HBEC3 cells, as no previous studies have examined this using a normal bronchial epithelial cell line model. We established an HBEC3 cell line, designated as HBEC3-BIN, that expresses mutant BRAFV600E in a doxycycline-regulated manner. Unlike our previous finding that KRASV12 upregulated both pERK and pAKT, mutant BRAFV600E upregulated pERK but not pAKT in HBEC3-BIN cells. Similar to KRASV12, BRAFV600E did not efficiently induce OIS. Interestingly, while BRAFV600E inhibited colony formation in anchorage-dependent conditions, it dramatically enhanced colony formation in anchorage-independent conditions in HBEC3-BIN. In HBEC3 cells without BRAFV600E or KRASV12 expression, p21 was only detected in the cytoplasm, and its localization was not altered by the expression of BRAFV600E or KRASV12. Next-generation sequencing analysis revealed an enrichment of gene sets known to be involved in carcinogenesis, including IL3/JAK/STAT3, IL2, STAT5, and the EMT pathway. Our results indicate that, unlike KRASV12, which promoted both, BRAFV600E enhances anchorage-independent growth but inhibits anchorage-dependent growth of HBEC3. This contrast may result from differences in activation signaling in the downstream pathways. Furthermore, HBEC3 cells appear to be inherently resistant to OIS, which may be partly due to the fact that p21 remains localized in the cytoplasm upon expression of BRAFV600E or KRASV12.

Abstract 199: Fluid shear stress enhances metastatic potential and rapidly alters metabolism of circulating tumor cells

Abstract Background and Purpose: Circulating tumor cells (CTCs) are exposed to mechanical and biochemical stresses including fluid shear stress (FSS) and oxidative distress. Recently we have shown that viable CTCs actively resist destruction when exposed to FSS by a mechano-adaptive mechanism that depends on RhoA-actomyosin activity [PMID: 32187555]. Since the RhoA-actomyosin axis is implicated in other cellular behaviors that might contribute to metastasis; herein, we explored the hypothesis that exposure to FSS alters the biology of viable CTCs to promote metastatic colonization. Methodology: To determine if exposure to FSS alters metastatic potential, we exposed PC-3 prostate and MDA-MB-231 breast cancer cells to FSS prior to injection into the tail-vein of NCG mice. We then monitored metastatic colonization by weekly bioluminescence imaging. Further, we examined if FSS exposure altered the invasive potential of PC-3 and MDA-MB-231 cells, as well as their ability to survive/proliferate under anchorage-independent conditions. We conducted GC/LC-MS metabolomic profiling and measured lactate production following FSS exposure. To investigate oxidative burden on FSS exposed cells, we used dihydroethidium to measure general ROS production and a lipid peroxide sensor. Results: We found that FSS exposure shortens the time for metastatic colonization for both cell lines. We found that FSS exposure leads to approximately two-fold increase in invasion through collagen matrix, which, for PC-3 cells, is RhoA dependent. Moreover, FSS exposure resulted in increased proliferation (~25%) under anchorage independent conditions in both cell lines. Metabolomic profiling revealed that FSS exposure resulted increased glycolysis, reduced entry into the TCA cycle, and increased lactate levels. We blocked glycolysis in PC-3 and MDA-MB-231 cells with 2-deoxyglucose and found that this increased the fraction of cells destroyed by FSS ~20-30%. We also investigated redox stress and found that compared to cells held in suspension, those exposed to FSS demonstrate lower ROS burden. Interestingly, cells exposed to FSS exhibit lower levels of lipid peroxides in a manner that depends on RhoA. Conclusion: Our findings demonstrate that brief pulses of FSS exposure can enhance metastatic potential, increase invasive and anchorage-independent proliferative capacity and rapidly alter metabolism. The phenotypic changes driven by exposure to FSS depend in part on RhoA activation but may also reflect other mechanisms by which cancer cells sense and respond to FSS. The rapid changes in metabolism may act to reduce oxidative distress while CTCs are in suspension. In summary, our data indicate that FSS exposure in the circulation not only represents a physical force that CTCs must overcome to survive, but it also provides instructive cues that may enhance the metastatic behavior of some CTCs. Citation Format: Devon L. Moose, Amanda N. Pope, Marion Dykstra, Eric B. Taylor, Patrick Breheny, Michael D. Henry. Fluid shear stress enhances metastatic potential and rapidly alters metabolism of circulating tumor cells [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 199.

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

Abstract B093: Investigating early events of ovarian cancer transcoelomic metastasis identifies the small Rho-GTPase RHOV as an essential gene for cellular detachment and aggregate formation

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.

Canonical Kaiso target genes define a functional signature that associates with breast cancer survival and the invasive lobular carcinoma histological type.

Invasive lobular carcinoma (ILC) is a low- to intermediate-grade histological breast cancer type caused by mutational inactivation of E-cadherin function, resulting in the acquisition of anchorage independence (anoikis resistance). Most ILC cases express estrogen receptors, but options are limited in relapsed endocrine-refractory disease as ILC tends to be less responsive to standard chemotherapy. Moreover, ILC can relapse after >15 years, an event that currently cannot be predicted. E-cadherin inactivation leads to p120-catenin-dependent relief of the transcriptional repressor Kaiso (ZBTB33) and activation of canonical Kaiso target genes. Here, we examined whether an anchorage-independent and ILC-specific transcriptional program correlated with clinical parameters in breast cancer. Based on the presence of a canonical Kaiso-binding consensus sequence (cKBS) in the promoters of genes that are upregulated under anchorage-independent conditions, we defined an ILC-specific anoikis resistance transcriptome (ART). Converting the ART genes into human orthologs and adding published Kaiso target genes resulted in the Kaiso-specific ART (KART) 33-gene signature, used subsequently to study correlations with histological and clinical variables in primary breast cancer. Using publicly available data for ERPOS Her2NEG breast cancer, we found that expression of KART was positively associated with the histological ILC breast cancer type (p < 2.7E-07). KART expression associated with younger patients in all invasive breast cancers and smaller tumors in invasive ductal carcinoma of no special type (IDC-NST) (<2 cm, p < 6.3E-10). We observed associations with favorable long-term prognosis in both ILC (hazard ratio [HR] = 0.51, 95% CI = 0.29-0.91, p < 3.4E-02) and IDC-NST (HR = 0.79, 95% CI = 0.66-0.93, p < 1.2E-04). Our analysis thus defines a new mRNA expression signature for human breast cancer based on canonical Kaiso target genes that are upregulated in E-cadherin deficient ILC. The KART signature may enable a deeper understanding of ILC biology and etiology. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

AR activates YAP/TAZ differentially in prostate cancer.

The Hippo signalling pathway is a master regulator of cell growth, proliferation, and cancer. The transcriptional coregulators of the Hippo pathway, YAP and TAZ, are central in various cancers. However, how YAP and TAZ get activated in most types of cancers is not well understood. Here, we show that androgens activate YAP/TAZ via the androgen receptor (AR) in prostate cancer (PCa), and that this activation is differential. AR regulates YAP translation while inducing transcription of the TAZ encoding gene, WWTR1 Furthermore, we show that AR-mediated YAP/TAZ activation is regulated by the RhoA GTPases transcriptional mediator, serum response factor (SRF). Importantly, in prostate cancer patients, SRF expression positively correlates with TAZ and the YAP/TAZ target genes CYR61 and CTGF We demonstrate that YAP/TAZ are not essential for sustaining AR activity, however, targeting YAP/TAZ or SRF sensitize PCa cells to AR inhibition in anchorage-independent growth conditions. Our findings dissect the cellular roles of YAP, TAZ, and SRF in prostate cancer cells. Our data emphasize the interplay between these transcriptional regulators and their roles in prostate tumorigenesis and highlight how these insights might be exploited therapeutically.

Abstract 6032: Exploiting metabolic vulnerabilities of ovarian cancer with upregulated succinate dehydrogenase

Abstract Ovarian cancer remains the deadliest of all gynecologic malignancies nationwide due to limited therapeutic options and chemotherapy resistance. The response to chemotherapy is substantially influenced by the high metabolic flexibility of ovarian cancer cells, which allows the cells to reprogram their metabolism and survive the treatment. Better understanding mechanisms regulating ovarian tumor metabolism could be exploited to develop new improved therapies. Our work demonstrated that the upregulation of mitochondrial enzyme succinate dehydrogenase (SDHA) is particularly prevalent (~20% of patients) in ovarian carcinoma and contributes to high energy tumor metabolism and cell survival. We observed that SDHA overexpressing ovarian cancer cells showed improved ability to survive and generate colonies in anchorage-independent conditions, which is an important feature of ovarian tumor cells surviving and spreading in peritoneal fluid (ascites). Proteomics data revealed that the SDHA overexpressing tumor cells showed reduced levels of proteins promoting anaerobic glycolysis (LDHA and HIF1α), and increased levels of mitochondrial pyruvate carrier (MPC) proteins. In addition, our tracer-based metabolomics data revealed that the elevated SDHA stimulates glutaminolysis by increasing mitochondrial glutamine supply fueling the TCA cycle with essential metabolites. We also demonstrated that the overexpression of SDHA is associated with a significant increase of OXPHOS and ATP production rate. Further, we reasoned that the SDHA overexpressing tumor cells could be particularly vulnerable to drugs disrupting glucose and/or glutamine metabolism. We performed a drug screening and identified an anti-metabolic compound shikonin known to disrupt glucose and amino acid metabolism. In in vitro studies, shikonin exhibited a profound anti-tumor efficacy and selectivity towards SDHA overexpressing tumor cells superior to that observed with chemotherapy. Importantly, our in vivo study validated the in vitro observations, and showed that shikonin is highly effective in suppressing SDHA overexpressing patient-derived tumor growth. In summary, the unique metabolic state of ovarian cancer associated with SDHA amplification could be successfully targetable offering a potential new treatment strategy for ovarian cancer patients. Citation Format: Lin Wang, Magdalena Bulicz, Patryk Mucha, Michael Kinter, Atul Pranay, Timothy M. Griffin, Benjamin F. Miller, Magdalena Bieniasz. Exploiting metabolic vulnerabilities of ovarian cancer with upregulated succinate dehydrogenase [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 6032.

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.

Abstract B047: Fluid shear stress enhances the metastatic potential and rapidly alters metabolism of circulating tumor cells

Abstract Background and Purpose: Circulating tumor cells (CTCs) are exposed to mechanical and biochemical stresses including fluid shear stress (FSS), due to blood flow, and oxidative distress, from a lack of matrix attachment. Recently we have shown that viable CTCs actively resist destruction when exposed to FSS by a mechano-adaptive mechanism that depends on RhoA-actomyosin activity (PMID: 32187555). Since the RhoA-actomyosin axis is implicated in other cellular behaviors that might contribute to metastasis, here we explored the hypothesis that exposure to FSS alters the biology of viable CTCs to promote metastatic colonization. Methodology: To determine if exposure to FSS alters metastatic potential, we exposed PC-3 prostate cells to FSS prior to injection into the tail-vein of NCG mice. We then monitored metastasis formation by weekly bioluminescence imaging. We also examined whether FSS exposure altered the ability of PC-3 and MDA-MB-231 cells to invade through a collagen I matrix, as well as their ability to survive/proliferate under anchorage-independent conditions. We conducted GC/LC-MS metabolomic profiling immediately after FSS exposure. To investigate oxidative burden on FSS exposed cells, we used dihydroethidium (10µM, 30 minutes) to measure general ROS production and a lipid peroxide sensor (5µM, 20 minutes). Results: We found that FSS exposure shortens the time for metastasis formation by 20% (median of 42 vs 52.5 days metastasis free; p=0.0222, log-rank). For both PC-3 and MDA-MB-231 cells we found that FSS exposure leads to ~2x increase in invasion through collagen matrix, and that increase depends on RhoA. Moreover, FSS exposure resulted in increased proliferation (~25%) under anchorage independent conditions in both cell lines. Metabolomic profiling revealed that FSS exposure resulted increased glycolysis, reduced entry into the TCA cycle, and a decrease in the metabolites that feed the folate cycle. We blocked glycolysis in PC-3 and MDA-MB-231 cells with 2-deoxyglucose (25mM, 2 hours) and found that this increased the fraction of cells destroyed by FSS ~20-30%. We also investigated redox stress and found that compared to cells held in suspension, those exposed to FSS demonstrate lower ROS burden. Interestingly, cells exposed to FSS exhibit lower levels of lipid peroxides in a manner that depends on RhoA. Conclusion: Our findings demonstrate that brief pulses of FSS exposure can enhance metastatic potential, increase invasive and anchorage-independent proliferative capacity and rapidly alter metabolism. The phenotypic changes driven by exposure to FSS depend in part on RhoA activation but may also reflect other mechanisms by which cancer cells sense and respond to FSS. FSS rapidly alters cellular metabolism in a manner that may act to reduce oxidative distress while CTCs are in suspension. In summary, our data indicate that FSS exposure in the circulation not only represents a physical force that CTCs must overcome to survive, but it also provides instructive cues that may enhance the metastatic behavior of some CTCs. This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR006) of the Conference Proceedings. Citation Format: Devon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. HenryDevon L. Moose, Amanda N. Pope, Marion Vanneste, Patrick Breheny, Eric B. Taylor, Michael D. Henry. Fluid shear stress enhances the metastatic potential and rapidly alters metabolism of circulating tumor 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 B047.

Cytomorphology and Gene Expression Signatures of Anchorage-independent Aggregations of Oral Cancer Cells.

Cancer cells with high anchorage independence can survive and proliferate in the absence of adhesion to the extracellular matrix. Under anchorage-independent conditions, cancer cells adhere to each other and form aggregates to overcome various stresses. In this study, we investigated the cytomorphology and gene expression signatures of oral cancer cell aggregates. Two oral cancer-derived cell lines, SAS and HSC-3 cells, were cultured in a low-attachment plate and their cytomorphologies were observed. The transcriptome between attached and detached SAS cells was examined using gene expression microarrays. Subsequently, gene enrichment analysis and Ingenuity Pathway Analysis were performed. Gene expression changes under attached, detached, and re-attached conditions were measured via RT-qPCR. While SAS cells formed multiple round-shaped aggregates, HSC-3 cells, which had lower anchorage independence, did not form aggregates efficiently. Each SAS cell in the aggregate was linked by desmosomes and tight junctions. Comparative transcriptomic analysis revealed 1,698 differentially expressed genes (DEGs) between attached and detached SAS cells. The DEGs were associated with various functions and processes, including cell adhesion. Moreover, under the detached condition, the expression of some epithelial genes (DSC3, DSP, CLDN1 and OCLN) were up-regulated. The changes in both cytomorphology and epithelial gene expression under the detached condition overall returned to their original ones when cells re-attached. The results suggest specific cytomorphological and gene expression changes in oral cancer cell aggregates. Our findings provide insights into the mechanisms underlying anchorage-independent oral cancer cell aggregation and reveal previously unknown potential diagnostic and therapeutic molecules.

L1CAM is required for early dissemination of fallopian tube carcinoma precursors to the ovary

Most ovarian high-grade serous carcinomas (HGSC) arise from Serous Tubal Intraepithelial Carcinoma (STIC) lesions in the distal end of the fallopian tube (FT). Formation of STIC lesions from FT secretory cells leads to seeding of the ovarian surface, with rapid tumor dissemination to other abdominal structures thereafter. It remains unclear how nascent malignant cells leave the FT to colonize the ovary. This report provides evidence that the L1 cell adhesion molecule (L1CAM) contributes to the ability of transformed FT secretory cells (FTSEC) to detach from the tube, survive under anchorage-independent conditions, and seed the ovarian surface. L1CAM was highly expressed on the apical cells of STIC lesions and contributed to ovarian colonization by upregulating integrins and fibronectin in malignant cells and activating the AKT and ERK pathways. These changes increased cell survival under ultra-low attachment conditions that mimic transit from the FT to the ovary. To study dissemination to the ovary, we developed a tumor-ovary co-culture model. We showed that L1CAM expression was important for FT cells to invade the ovary as a cohesive group. Our results indicate that in the early stages of HGSC development, transformed FTSECs disseminate from the FT to the ovary in a L1CAM-dependent manner.

Phytochemicals from Vanda bensonii and Their Bioactivities to Inhibit Growth and Metastasis of Non-Small Cell Lung Cancer Cells

The most prevalent lung cancer is non-small cell lung cancer (NSCLC). This lung cancer type often develops other organ-specific metastases that are critical burdens in the treatment process. Orchid species in the genus Vanda have shown their potential in folkloric medication of diverse diseases but not all its species have been investigated, and little is known about their anticancer activities against NSCLC. Here, we firstly profiled the specialized metabolites of Vanda bensonii and examined their capability to inhibit growth and metastasis of NSCLC using NCI-H460 cells as a study model. Four phytochemicals, including phloretic acid methyl ester (1), cymbinodin-A (2), ephemeranthoquinone B (3), and protocatechuic acid (4), were isolated from the whole plant methanolic extract of V. bensonii. The most distinguished cytotoxic effect on NCI-H460 cells was observed in the treatments with crude methanolic extract and compound 2 with the half maximal inhibitory concentrations of 40.39 μg mL−1 and 50.82 μM, respectively. At non-cytotoxic doses (10 μg mL−1 or 10 μM), only compound 1 could significantly limit NCI-H460 cell proliferation when treated for 48 h, while others excluding compound 4 showed significant reduction in cell proliferation after treating for 72 h. Compound 1 also significantly decreased the migration rate of NCI-H460 cells examined through a wound-healing assay. Additionally, the crude extract and compound 1 strongly affected survival and growth of NCI-H460 cells under anchorage-independent conditions. Our findings proved that natural products from V. bensonii could be promising candidates for the future pharmacotherapy of NSCLC.

Abstract 5378: PMD-026, a first in class oral RSK inhibitor, demonstrates activity against hormone receptor positive breast cancer with acquired CDK4/6 inhibitor resistance

Abstract PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK, PDK-1 and PI3K pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Specifically, both MAPK and PI3K pathways are implicated in resistance to standard of care (SOC) CDK4/6 inhibitors in hormone receptor positive (HR+) breast cancer (BC). RSK2 is expressed in 80% of HR+ BC, therefore we hypothesized that disrupting these critical pathways with PMD-026 could overcome resistance to CDK4/6 inhibitors through cell growth inhibition, apoptosis induction and downstream target inhibition alone and in combination with SOC SERD fulvestrant. In preclinical studies and a Phase I/Ib clinical trial in metastatic BC, PMD-026 demonstrated a good safety profile, making it an ideal candidate for the CDK4/6 inhibitor resistant HR+ BC population. Here in we demonstrate that PMD-026 inhibits growth of HR+ BC cell lines MCF-7 and T47D in vitro at IC50 values ranging from 3.7 to 10.1 µM, resulting in dephosphorylation of the RSK downstream effector YB-1 and induction of apoptosis through PARP cleavage. Comparatively, the MAPK pathway remains active in the presence of fulvestrant, as indicated by the expression of pYB-1. We determined that PMD-026 synergizes with fulvestrant in the MCF-7 model in vitro with a combined drug index (CDI) of 0.73 to 0.92. The next step was to determine if this combination is effective in cells resistant to CDK4/6 inhibitors. For that purpose, MCF-7 and T47D cells were treated with increasing concentrations of the CDK4/6 inhibitor palbociclib (Ibrance) up to 2 µM for several months. The generated Ibrance resistant cell lines (MCF-7-IBR, T47D-IBR) are insensitive to Ibrance up to 2 µM, but their parental counterparts have IC50 values ranging from 0.05 to 0.15 µM. In addition, cross-resistance to an additional CDK4/6 inhibitor, abemaciclib (Verzenio), was observed in the resistant cell lines, but the parental lines remained sensitive at IC50 values ranging from 0.15 to 0.25 µM. Most importantly, both parental and resistant cell lines remained sensitive to PMD-026 at similar IC50 values, indicating that CDK4/6 inhibitor resistance does not affect PMD-026 sensitivity. PMD-026 was then tested in combination with fulvestrant in anchorage independent conditions in the MCF-7-IBR cells. It was determined that this combination was highly synergistic with CDI ranging from 0.55 to 0.79. Together, these data support the application of PMD-026 and fulvestrant as a novel method to stop the growth of HR+ BC with acquired resistance to CDK4/6 inhibitors. Additionally, given that the MAPK pathway is upregulated in response to PI3K inhibition, the PMD-026 and fulvestrant combination is a potential alternative treatment for HR+ BC patients. Citation Format: Aarthi Jayanthan, Lambert Yue, My-my Huynh, Gerrit Los, Sandra E. Dunn. PMD-026, a first in class oral RSK inhibitor, demonstrates activity against hormone receptor positive breast cancer with acquired CDK4/6 inhibitor resistance [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 5378.

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.

Abstract 3327: Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors

Abstract The non-receptor protein tyrosine phosphatase SHP2 (PTPN11) plays an important role in the regulation of RAS/MAPK signal transduction downstream of growth factor receptor activation. Loss of SHP2 activity suppresses tumor cell growth, making SHP2 a potential target for cancer therapy. Here we report the discovery of GDC-1971 (formerly RLY-1971), a highly potent, selective, and orally bioavailable small-molecule SHP2 inhibitor that stabilizes SHP2 in a closed, auto-inhibited conformation. GDC-1971 potently inhibits both wild-type SHP2 (IC50 &amp;lt;1nM) and the E76K activating mutant (IC50 &amp;lt;250nM) in biochemical assays. In standard 2-dimensional and anchorage-independent growth conditions, GDC-1971 inhibits cellular proliferation in models harboring receptor tyrosine kinases (RTKs), SHP2, NF1, KRAS, or BRAF mutations in a dose-dependent manner. GDC-1971 potently inhibits the proliferation of cellular models harboring KRAS G12C or G12A mutations (median IC50 &amp;lt;80 nM) compared to models harboring other KRAS G12, G13 or Q61 mutations (median IC50 &amp;gt;1 uM), indicating a link between KRAS GTP hydrolysis and SHP2 dependency. Despite this trend, some non-KRAS G12C or G12A cell lines harboring other KRAS mutations responded to GDC-1971 in vitro, suggesting some heterogeneity in RTK/SHP2 signaling dependence in subsets of other KRAS mutants. In vivo, GDC-1971 demonstrates dose-dependent RAS/MAPK pathway inhibition and induces significant tumor-growth inhibition in human xenograft models harboring EGFR and KRAS alterations at continuous daily doses that are well tolerated. Given the reported role of SHP2 as a critical mediator of resistance to targeted therapies, we assessed the activity of GDC-1971 combinations in multiple contexts. We observed increased suppression of the MAPK signaling cascade and anti-proliferation synergy when combining GDC-1971 with EGFR, ALK, and KRAS G12C inhibitors in vitro. The observed in vitro synergy translated to dramatic anti-tumor growth effects in vivo. GDC-1971 in combination with the KRAS G12C covalent inhibitor GDC-6036 resulted in significant regressions at doses well below those required for single agent activity in a KRAS G12C-mutant NSCLC xenograft model. In rodent and dog toxicology studies, GDC-1971 is well tolerated at exposures above those required to induce regression in xenograft models. The biochemical and cellular potency and favorable pharmaceutical properties of GDC-1971 support the further clinical development in RTK/MAPK pathway altered tumors using continuous daily dosing alone and in combination with other targeted agents, including the KRAS-G12C inhibitor GDC-6036 (clinical trial NCT04449874). Citation Format: Bret Williams, Alexander Taylor, Olivia Orozco, Christopher Owen, Elizabeth Kelley, Andre Lescarbeau, Kelley Shortsleeves, Randy Kipp, Vy Nguyen, Erin Brophy, Jeremy Wilbur, Yong Tang, David Lanzetta, Nigel Waters, Sherri Smith, Fabrizio Giordanetto, Paul Maragakis, Jack Greismann, Lindsay Willmore, Eric Therrien, Yang Xiao, Marie Evangelista, Luca Gerosa, Eva Lin, Mark Merchant, Alfonso Arrazate, Emily Chan, Pablo Sáenz-López Larrocha, Stefan Chun, Thomas Hunsaker, Gauri Deshmukh, Christine M. Bowman, David E. Shaw, Mark Murcko, Mahesh Padval, W Patrick Walters, James Watters, Donald A. Bergstrom. Discovery and characterization of the potent, allosteric SHP2 inhibitor GDC-1971 for the treatment of RTK/RAS driven tumors [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 3327.

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.

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.