Soft Agar Assay Research Articles

Echinatin induces reactive oxygen species-mediated apoptosis via JNK/p38 MAPK signaling pathway in colorectal cancer cells.

Colorectal cancer (CRC) is a very common and deadly cancer worldwide, and oxaliplatin is used as first-line chemotherapy. However, resistance usually develops, limiting treatment. Echinatin (Ech) is the main component of licorice and exhibits various therapeutic effects on inflammation-mediated diseases and cancer, ischemia/reperfusion, and liver injuries. The present study elucidated the underlying molecular mechanism of Ech-induced apoptosis in both oxaliplatin-sensitive (HT116 and HT29) and -resistant (HCT116-OxR and HT29-OxR) CRC cells. To evaluate the antiproliferative activities of Ech, we performed MTT and soft agar assays. Ech reduced viability, colony size, and numbers of CRC cells. The underlying molecular mechanisms were explored by various flow cytometry analyses. Ech-induced annexin-V stained cells, reactive oxygen species (ROS) generation, cell cycle arrest, JNK/p38 MAPK activation, endoplasmic reticulum (ER) stress, mitochondrial membrane potential depolarization, and multi-caspase activity. In addition apoptosis-, cell cycle-, and ER stress-related protein levels were confirmed by western blotting. Moreover, we verified ROS-mediated cell death by treatment with inhibitors such as N-acetyl-L-cysteine, SP600125, and SB203580. Taken together, Ech exhibits anticancer activity in oxaliplatin-sensitive and -resistant CRCs by inducing ROS-mediated apoptosis through the JNK/p38 MAPK signaling pathway. This is the first study to show that Ech has the potential to treat drug-resistant CRC, providing new directions for therapeutic strategies targeting drug-resistant CRC.

ICSBP-induced PD-L1 enhances osteosarcoma cell growth.

BackgroundInterferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor induced by IFN-γ. We previously reported that ICSBP expression promotes osteosarcoma progression by enhancing transforming growth factor-β signaling. In cancer cells, programmed death-ligand 1 (PD-L1) contributes to immune escape and may also be involved in tumor progression. Because IFN-γ induces the expression of both ICSBP and PD-L1, we explored the association between ICSBP and PD-L1 expression in terms of osteosarcoma progression.MethodsThree osteosarcoma cell lines (Saos2, U2OS, and 143B) were employed. Gene expression was measured by qRT-PCR, and protein levels were assessed by immunoblotting. PD-L1 expression was evaluated in cells overexpressing ICSBP and in ICSBP knockdown cells. The effects of PD-L1 expression on cell growth were examined by MTS assays, Incucyte analysis, soft agar assays, and three-dimensional (3D) culture. Cell cycle and apoptosis were evaluated by FACS analysis of cells stained with propidium iodide (PI) and annexin V/PI, respectively. The antitumor effects of PD-L1 knockdown without or with doxorubicin treatment were evaluated in vivo in nude mice bearing ICSBP-overexpressing 143B cell xenograft. The clinical relevance of PD-L1 and ICSBP expression was evaluated immunohistochemically using a human osteosarcoma microarray and through analysis of publicly available data using Gene Expression Profiling Interactive Analysis2.ResultsICSBP overexpression upregulated PD-L1 expression in all three cell lines, whereas ICSBP knockdown decreased the PD-L1 expression. PD-L1 knockdown attenuated the cell growth and reduced colony-forming capacity in both soft agar assays and 3D culture. PD-L1 knockdown increased apoptosis and induced G2/M arrest, which was associated with decreased expression of survivin, cyclin-dependent kinase 4 (CDK4), cyclin E, and cyclin D1 expression and increased the expression of p27, phosphorylated Cdc2, and phosphorylated Wee1. PD-L1 knockdown decreased the growth of tumor xenografts and increased the doxorubicin sensitivity of ICSBP-overexpressing 143B cells both in vitro and in vivo. PD-L1 was expressed in human osteosarcoma tissues, and its expression was moderately correlated with that of ICSBP in osteosarcoma patients.ConclusionICSBP regulates PD-L1 expression in osteosarcoma cells, and PD-L1 knockdown combined with doxorubicin treatment could represent a strategy for controlling osteosarcoma expressing ICSBP.

Chelerythrine Inhibits Stemness of Cancer Stem-Like Cells of Osteosarcoma and PI3K/AKT/mTOR Signal.

Chelerythrine (CHE) is widely found in many herbs and is the main alkaloid constituent of Toddalia asiatica (L.) LAM. It has been proved to exert remarkable antitumor, antifungal, anti-inflammatory, and antiparasitic effects. In osteosarcoma, CHE is reported to inhibit proliferation and promote apoptosis. However, the effect of CHE on cancer stem-like cells (CSCs), which contribute to metastasis and recurrence in osteosarcoma, is still largely unknown. In this study, we investigated the effects of CHE on the stemness and malignant behaviors of CSCs derived from osteosarcoma cells. CSCs were enriched by culturing in serum-free medium. The effects of CHE on stemness were measured by detecting stemness factors and sphere formation ability. The effects of CHE on chemosensitivity to doxorubicin and MTX were measured by Annexin V-FITC/PI double staining. The effects of CHE on CSC malignancy were measured by performing CCK-8, colony formation, tumor formation in soft agar, migration, and invasion assays. We first enriched CSCs from osteosarcoma cells, which were characterized by upregulated stemness markers, including Oct4, Nanog, and Nestin. The addition of CHE clearly decreased malignant behaviors, including colony formation, tumor formation in soft agar, migration, and invasion. CHE also inhibited stemness and thus induced the failure of sphere formation. Moreover, CHE promoted apoptosis induced by chemo agents, including doxorubicin (DOX) and methotrexate (MTX). After CHE treatment, the protein expression of MMP-2/9 was significantly decreased, potentially inhibiting invasion. CHE also exhibited an inhibitory effect on the phosphorylation of PI3K, AKT, and mTOR, which is an upstream regulatory signaling pathway of MMP-2/9. In summary, CSCs derived from U2OS and MG-63 cells, CHE could inhibit the stemness and malignant behaviors of CSCs potentially by inhibiting the PI3K/AKT/mTOR signaling pathway.

HMMR promotes peritoneal implantation of gastric cancer by increasing cell–cell interactions

BackgroundDistant metastasis is the prominent factor for cancer-induced death of gastric cancer in which peritoneum is one of the dominating targets of gastric cancer metastasis. However, there is still a lack of effective predictive indicators and treatment methods for gastric cancer patients with peritoneal metastasis.MethodsA clustering assay was used to investigate the cell aggregates formation ability. While the soft agar assay and anoikis assay were performed to detect the anchorage-independent growth and anoikis-resistant ability respectively. Luciferase activity assay, western blotting and immunofluorescence were used to explore the effect of HMMR on AKT signaling activity. The peritoneal implantation model was examined to explore the role of HMMR in vivo.ResultsSilencing of HMMR expression markedly reduced the peritoneal metastasis of gastric cancer cells through reducing cell–cell interactions. Mechanistically, HA-HMMR could activate Akt signaling, thus succeeding in distant colonization and metastatic outgrowth. Importantly, inducible depletion of HMMR significantly abrogates peritoneal implantation of gastric cancer in vitro and in vivo.ConclusionOur study highlights that HMMR promotes peritoneal implantation of gastric cancer. A better understanding of HMMR’s functions and mechanism might provide a novel therapeutic target and prognostic marker for metastatic gastric cancer.

Potential mechanisms underlying the promoting effects of 3D collagen scaffold culture on stemness and drug resistance of glioma cells

Background3D collagen scaffold culture is a good tool to study glioma metastasis and recurrence in vitro. MethodsThe effect of 3D collagen culture on the colony formation, the sphere formation, and drug sensitivity of glioma cells was observed by soft-agar colony formation assays, sphere formation assays, and CCK-8 assays, respectively. 3D-glioma-drug genes were identified by previous results and online databases. Gene enrichment and PPI analyses were performed by R software and Metacsape. Hub 3D-glioma-drug genes were screened by STRING and Cytoscape. TCGA and CGGA databases and R software were used to analyze the distribution of hub genes in glioma and their effects on the prognosis. Western Blot was used to verify the effect of 3D collagen culture on the expression of hub genes. miRNAs targeting hub genes were predicted by ENCORI. Results3D collagen scaffold culture promoted colony formation, sphere formation, and drug resistance of glioma cells. There were 77 3D-glioma-drug genes screened, and the pathways enriched in the protein interaction network mainly included responses to stressors, DNA damage and repair, and drug metabolism. Hub 3D-glioma-drug genes were AKT1, ATM, CASP3, CCND1, EGFR, PARP1, and TP53. These genes and predicted miRNAs were expressed differentially in glioma samples and partially affected the prognosis of patients with glioma. These findings suggested these hub genes and miRNAs may play a key role in the effects generated by the 3D culture model and become new markers for glioma diagnosis and treatment.

NCAPG promotes tumorigenesis of bladder cancer through NF-κB signaling pathway

The non-SMC condensin I complex subunit G (NCAPG) is a subunit of the condensin complex, many studies have shown that NCAPG is aberrantly expressed in different tumors and closely associated with poor prognosis, but its role in bladder cancer is unclear. In this paper, we found that NCAPG expression was upregulated in bladder cancer in tumor-related databases, and further verified the expression of NCAPG in bladder cancer tissues as well as bladder cancer cell lines by tissue microarray, qPCR, and WB. Next, we explored the changes in bladder cancer cell proliferation as well as migration after NCAPG knockdown by cell growth curve, colony formation, soft agar assay, and xenograft model. Finally, we examined the changes in downstream signaling pathways after NCAPG knockdown using RNA-Seq, and we found that the NF-κB signaling pathway was inhibited with NCAPG gene knockdown, which was verified by luciferase reporter assay as well as WB. In conclusion, our results illustrate that NCAPG knockdown can inhibit the proliferation of bladder cancer cells through the NF-κB signaling pathway. This finding demonstrates that NCAPG could be a potential target for the treatment of bladder cancer.

BRD9 is an essential regulator of glycolysis that creates an epigenetic vulnerability in colon adenocarcinoma.

The intensive interplay between aberrant epigenetic events and metabolic remodeling represents one of the hallmarks of tumors, including colon cancer. The functions of Bromodomain Containing Protein BRD-9 in colon cancer remains indefinite. We aimed to identify the biological roles and clinical significance of BRD9 in colon cancer. The univariate- and multi-variate Cox regression models were used to screen risk epigenetic regulators. Kaplan-Meier analysis and Pearson correlation analysis were used to assess clinical significance of BRD9. CCK-8 assays, colony formation assay, Transwell, and soft-agar assay were performed to determine the in vitro roles of BRD9. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of colon cancer cells were evaluated by a Seahorse XF Extracellular Flux Analyzer. In vivo models and RT-qPCR, western blotting, and Chromatin Immunoprecipitation (ChIP) assay were conducted to explore the functional roles of BRD9 in COAD. In the study, we detected the expressions of 662 epigenetic regulators in COAD and identified a series of 42 hazard epigenetic factors with p < 0.05. Low-throughput MTT assays highlighted that BRD9 is an essential target, and targeting BRD9 could reduce significant decreases of cell growth. BRD9 overexpression could notably elevate proliferation and migration potentialities, whereas, BRD9 ablation abolished these effects. Mechanistically, functional enrichment analysis indicated the potential associations between BRD9 and glycolysis metabolism. In addition, BRD9 epigenetically coordinates the H3K27ac modifications on the promoter regions of ENO2 and ALDOC, inducing enhanced glycolysis activity. Lastly, I-BRD9 could significantly suppress the growth of colon cancer cells in vitro and in vivo. Together, our study revealed previously unidentified roles of BRD9 in colon cancer metabolism and tumor progression, indicating that BRD9 could be a valuable therapeutic target for COAD patients.

Abstract 861: Nuclear translocation of Axl during early breast cancer progression

Abstract Axl is a receptor tyrosine kinase (RTK) that is activated by its ligand Gas6 and regulates proliferation, cell survival, and clearance of apoptotic cells in both normal physiology and a number of disease states. In breast cancer, Axl is overexpressed in Her2+ and triple-negative breast cancers and is associated with resistance to RTK targeted therapies. Recent studies have shown that upon kinase inhibition, the Axl intracellular domain (ICD) can translocate to the nucleus, however, the biological significance and functions for nuclear Axl are poorly understood. Furthermore, a functional role for Axl during ductal carcinoma in situ (DCIS) progression, a requisite step for invasive breast cancer, has not been established. To address whether Axl mediates localized invasion during DCIS progression, Axl was deleted in a DCIS cell line (MCF10A-DCIS.com) using a CRISPR-Cas9 approach. Deletion of Axl shows a significant decrease in progression to invasive cancer in vivo, which is accompanied by decreased proliferation, however microinvasion is unchanged as compared to wildtype. Analysis of patient samples of pure DCIS, DCIS with microinvasion, and DCIS with invasive cancer shows a significant increase in Axl expression in patients with evidence of invasion. Interestingly, there is a significant increase in nuclear Axl in all patient samples with evidence of invasion as compared to normal breast or pure DCIS, suggesting an important role for Axl-ICD in progression to invasive cancer. Soft agar assays using normal cells (MCF10A) shows that while full length Axl (Axl-FL) is sufficient to induce colonies, Axl-ICD-transduced cells do not form colonies, suggesting that Axl-ICD alone does not have oncogenic activity. However, Axl-ICD significantly increases colonies in DCIS cells, suggesting that the ICD is important in the progression of transformed cells. Ongoing studies are aimed at identifying transcriptional targets of Axl-ICD in DCIS cells. These studies will advance our understanding of how Axl mediates breast cancer progression and will have critical implications for ongoing therapeutic strategies aimed at targeting Axl. This work is supported by NIH R01CA212518 (H.L.M.) and Susan G. Komen CCR1637765 (H.L.M.). Citation Format: Darby R. Graham, Kelly D. Hebert, Emma E. Newton, Angelica M. Gomes, Fariba Behbod, Heather L. Machado. Nuclear translocation of Axl during early breast cancer progression [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 861.

Abstract 3002: Characterization of novel role for Rab27B in autophagy regulation in colorectal cancer

Abstract Autophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a well-known regulator of vesicle trafficking and exosome secretion, also regulates autophagy process. Depletion of Rab27B in colorectal cancer (CRC) cells by CRISPR-Cas9 and siRNA resulted in an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62 indicating a defect in the autophagy flux. To characterize this autophagy defect, we used EGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining and identified that the degradation step of the autophagy flux is inhibited when Rab27B is silenced. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that autophagy defect resulting from Rab27B deletion will cause reduced stress response and tumor growth. Indeed, Rab27B knockout resulted in a 60% reduction in cell viability in response to starvation and a 94% reduction in colony formation in soft agar assay. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells HCT116 and observed that Rab27B deletion resulted in a significant reduction of tumor size (p&amp;lt;0.0001). Next, to analyze the therapeutic potential of Rab27B inhibition in CRC, we sought to identify small-molecule inhibitors of Rab27B GTPase activity in vitro through high-throughput drug screening from a panel of ∼2,100 compounds contained within the KU-HTSL chemical library. This screening resulted in the identification of three compounds (BTB00809, BTB03584, and KM01532) that demonstrated Rab27B GTPase activity inhibition. Interestingly, inhibition of Rab27B in CRC cells with the compound km01532 resulted in a similar increase of LC3-II in CRC cells HCT116 and SW480 as seen in the Rab27B knockout. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on investigating the mechanism of how Rab27B functions in the autophagy process, and whether Rab27B can be targeted as a potential therapeutic strategy for CRC using the small molecule compound km01532. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan Dixon. Characterization of novel role for Rab27B in autophagy regulation in colorectal 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 3002.

Abstract 1048: Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach

Abstract Background: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. EWS/FLI interacts with lysine-specific demethylase 1 (LSD1) that is highly expressed in pediatric sarcomas, to repress critical tumor suppressors. We have developed a potent reversible LSD1 inhibitor, SP-2577 (Seclidemstat), that shows the ability to impair tissue culture cell viability in multiple ES cell lines and is currently in clinical trials. A more favorable therapeutic response to SP-2577 may be obtained by combining drugs that target multiple pathways and/or inhibit resistance mechanisms. Experimental Design: In this study, we applied an arrayed CRISPR screening (Synthego) to identify survival genes that can be candidates for molecularly targeted drugs. Gene candidates were validated using western blot, qPCR, soft agar assay. Ewing’s Sarcoma A673 ABL2i knockdown and scramble cell lines were generated, implanted into nude mice, and treated with SP-2577. Lastly, combinational drug treatments using FDA-approved inhibitors with SP-2577 were tested in 2D and 3D cultures. Results: CRISPR druggable library screen identified that downregulation of several genes resulted in increased SP-2577 cytotoxicity in multiple ES cell lines. Importantly, ABL2 knockdown was found to have a significant synergy in combination with SP-2577 treatment, both in vitro and in vivo. siRNA knockdown of ABL2 in combination with SP-2577 treatment revealed an increase in JNK signaling and Cleaved Caspases-3 activation with decreased Src phosphorylation. This data suggests that downregulation of ABL2 in combination with SP-2577 treatment promotes activation of apoptosis pathways in ES. In addition, combination treatment of SP-2577 with drugs targeting ABL2 pathway showed high synergy in vitro. Conclusion: This study highlights the potential for a combination treatment of SP-2577 with ABL2 inhibitors in ES patients. Further, our observations support the capability of arrayed CRISPR screening to identify new therapeutic strategies in ES. Citation Format: Samuel Sampson, Trason Thode, Kevin Drenner, Ryan Rodriguez Del Villar, Annika Ozols, Mohan Kaadige, Alexis Weston, Serina Ng, Tithi Ghosh Halder, Shelby Rheinschmidt, David Anderson, Kevin Holden, Raffaella Soldi, Sunil Sharma. Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach [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 1048.

Abstract LB060: Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST)

Abstract Background: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas of the peripheral nervous system. Loss of function of the polycomb repressive complex 2 (PRC2) have been identified in up to 80% of MPNST cases, via recurrent genetic mutations in the core components, EED and/or SUZ12. However, the transcriptional mis-regulation incurred by the loss of transcriptional repression in MPNST, through loss of H3K27me3, has yet to be fully deciphered. We hypothesize that PRC2 loss leads to transcriptional regulatory imbalance between key epigenetic regulating complexes in this malignancy, which may contribute to the oncogenesis of this disease. Therefore, this project aims to elucidate the role of the SWI/SNF chromatin remodeling complexes in MPNST, epigenetic regulators known to act antagonistically to PRC2. Method: This project uses a CRISPR knock out screen combined with a single cell RNA sequencing readout to target 44 known components of the SWI/SNF complexes. This technology enables large scale phenotypic assays, which we leverage to decipher the complicated epigenetic regulatory network within these lesions. Resulting genes of interest were further studied via loss-of-function assays coupled with colony formation assay and chromatin immunoprecipitation DNA sequencing (ChIP-seq) experiments. Results: This CRISPR screen highlighted core SWI/SNF components, SMARCA4 and SMARCE1, as genes of interest in MPNST. Knockdown of these genes in MPNST cell lines reduced proliferation, indicating a role for these genes in MPNST growth and viability. Further, investigations via soft agar assay found both SMARCA4 and SMARCE1 knock out to decrease the tumorigenic potential of MPNST cells. The potential dependency of SMARCA4 function in MPNST cells on PRC2 mutation was investigated using a doxycycline-inducible PRC2-restoration MPNST cell line model. ChIP-seq results suggest that loss of PRC2 does not substantially affect the binding of SMARCA4-containing SWI/SNF complexes across the genome. DPF1, a member of the neuronal-specific SWI/SNF chromatin remodeling complex, was further highlighted as a unique regulator of MPNST transcription via the CRISPR single-cell screen. CRISPR knockdown of this gene in MPNST cell lines has been found to decrease proliferation, while soft agar assay experiments highlighted that DPF1 loss reduces cell tumorigenicity. The unique dependency of this gene observed in MPNST but not in any other cancer subtypes highlights the potential of DPF1 to be a novel and unique therapeutic target in MPNST. Conclusion: Thus far, our studies have shown that members of the SWI/SNF complex play key roles in the MPNST cell proliferation and tumorigenicity. The function of these complexes in MPNST, however, may be independent of the transcriptional imbalance caused by PRC2 mutation. Citation Format: Béga Murray, Xiyuan Zhang, Shahroze Abbas, Hilda Jafarah, Jack F. Shern. Single-cell CRISPR screen of malignant peripheral nerve sheath tumors (MPNST) [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 LB060.

Abstract 6034: Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments

Abstract Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common malignancy diagnosed in Caucasians with an incidence of about one million cases per year. TP53 and p16 are the most common mutated genes in cSCC. Previous studies of cSCC have shown that patients with both p53 and p16 mutations have lower survival rates. There are currently no syngeneic mouse preclinical models to accelerate the efficacy of immunotherapy combinations to treat cSCC. Our laboratory has generated several genetically engineered mouse models (GEMMs) containing tissue-specific p53 and p16 gene mutations. Recently, we have immortalized three murine cSCC cell lines (cSCC-5690, cSCC-8006, and cSCC-8271) derived from spontaneous skin tumors from GEMMs. Therefore, we hypothesized that the double mutant cell line cSCC-5690 enhances cell survival, tumor aggressiveness, and has lower sensitivity to antibody immunotherapy. Protein and RNA expression of the cell lines were validated by western blotting and qRT-PCR assays. Sphere and soft agar assays were used to evaluate the stem cell population and tumorigenicity of the cell lines. Then, we examined the chemosensitivity with cisplatin through clonogenic assays. To further study the tumorigenicity of cells, we evaluated in vivo tumor growth on C57BL/6J immunocompetent mice by injecting subcutaneously the tumor cell lines. The three cell lines expressed different levels of keratin 14 (K14), but cSCC 5690 and 8006 expressed high levels of genes related to oncogenic pathways as well as mesenchymal markers. The cell line cSCC-5690 expressing mutant p53 R172H with p16 gene deletion, showed an enhanced sphere, and colony tumor formation and both tumorigenic cell lines (cSCC-5690, 8006) showed different sensitivities to cisplatin treatment. A higher tumor growth rate was observed with cSCC-5690 compared to cSCC-8006, while cSCC-8271 showed no tumor growth. Immunohistochemistry studies demonstrated that both cSCC-5690 and 8006 tumors expressed vimentin but only cSCC-5690 expressed K14. The most aggressive syngeneic tumor model (cSCC-5690) was used to evaluate the anti-tumor efficiency of anti-PD1, anti-CD47 antibodies, and STING-agonist. Preliminary results revealed a significant tumor response only to STING agonist (c-di-GMP) which activates the innate and adaptive immune response. Therefore, we were able to characterize all three cell lines and established a new mouse model of cSCC as a relevant platform for immunotherapy studies. Citation Format: Alanis Enid Rodriguez Rosario, Roberto Rangel, Jeffrey Myers. Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments [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 6034.

Abstract 1669: Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation

Abstract Ewing sarcoma (EwS), an aggressive bone and soft tissue cancer predominantly occurring during adolescence, is driven by the chimeric EWS-FLI1 oncogene. It is assumed that a permissive cellular context or potentially additional complementing mutations are necessary for the EWS-FLI1 fusion to exert its full oncogenic effect. Prior efforts to generate genetic EwS mouse models failed because of high EWS-FLI1 induced apoptosis when expressed in various candidate progenitor cell types. In our study, we restricted EWS-FLI1 expression to mesenchymal stem cells during endochondral bone formation by crossing a mouse containing EWS-FLI1 knocked into the Rosa26 locus with a mouse expressing Prx1-driven Cre recombinase. However, the resulting EFprx1 progeny died after birth as functional consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EWS-FLI1 transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but not fully transformed. However, we observed elevated levels of cleaved PARP-1, a surrogate marker of apoptosis. This apparent increased cellular turnover was paralleled by down-regulation of anti-apoptotic Bcl2a family genes as identified by RNA-seq analysis of EFprx1 MSCLC. The peak incidence of human EwS occurs during puberty, a developmental period in which several growth promoting hormones are highly enriched in the bone microenvironment. Among them is IGF1, which is known for its anti-apoptotic activity through upregulation of Bcl2 family genes. As previous studies implied IGF1 signaling essential in EwS pathogenesis, we speculated that IGF1 treatment may aid in the transformation process of EFprx1 MSCLC. In fact, a significant increase in soft-agar colony formation was observed upon IGF1 treatment for 4 weeks. To test whether IGF1 had irreversibly reprogrammed EFPrx1 MSCLC, we picked colonies grown in presence of IGF1, expanded and re-assayed them after 5 weeks in absence of the hormone. Strikingly, EFprx1 MSCLC retained their transformed phenotype not only in the soft-agar assay but also upon xenotransplantation in mice. Gene set enrichment analysis of upregulated genes from these stably hormone-reprogramed fully transformed EFprx1 MSCLC showed a significant enrichment for the EWS-FLI1 specific transcriptional signature and cell cycle-related pathways, while apoptotic gene sets were downregulated. Our data suggest that transient IGF1 exposure in the bone microenvironment may be required for EWS-FLI1 driven transformation. We propose that EwS formation could be influenced by hormonal changes during puberty. Based on these findings, we postulate that correct developmental timing of EWS-FLI1 expression may play a pivotal role for the generation of an EwS mouse model. Citation Format: Rahil Noorizadeh, Florian Halbritter, Barbara Sax, Wolfgang Mikulits, Richard Moriggl, Heinrich Kovar. Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation [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 1669.

Abstract 6376: Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells

Abstract Background: Many studies have suggested that MET protein overexpression or MET amplification plays a critical role in the progression of gastro-esophageal cancer (GC) and negatively affects survival in patients with GC. However, the criteria used to define overexpression of MET protein have differed among many studies, and in contrast to the initial phase II studies, the phase III trials failed to show any clinical benefit from anti-MET therapies in GCs, even in patients with MET-positive disease. Several studies have used consistent criteria to define MET amplification on FISH, and recent MET inhibitor trials for MET amplified cancers have been successful. Furthermore, hyperactivation of MET pathway are known to reduce the growth-inhibitory effect of trastuzumab in HER2 positive GC. Currently, there is no FDA approved MET inhibitors for GC. Capmatinib and savolitinib, which are selective MET inhibitors, has been evaluated for various MET positive cancers. The aim of this study was to test these MET inhibitors on MET amplified gastro-esophageal cancer (GC) cells. Material and Methods: Among a total of 37 GC cells in our lab, the following cells were positive for MET by copy number amplification (CNV &amp;gt; 10): SNU-620, ESO51, MKN-45, SNU-5 and OE-33. We assessed cytotoxic response of these GC cells to capmatinib or savolitinib alone by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. Western blots were performed to assess the cellular activity of capmatinib or savolitinib in MET signal pathway. Xenograft studies were performed to evaluate in vivo therapeutic efficacy of savolitinib with MKN-45 cells. Results: Savolitinib and capmatinib showed anti-proliferative activity against MET amplified GC cell lines in dose-dependent manner. There was no inhibitory effect on GC cells without MET amplification, such as SNU-216 and SNU-1750. Savolitinib inhibited phosphorylation of MET and down-stream signaling pathway, such as AKT or ERK, in MET amplified GC cells. We observed that treatment with savolitinib induced a significant decrease in the number of colonies formed in soft agar. In addition, Savolitinib exhibited dose-responsive anti-tumor efficacy in xenograft model with MKN-45 GC cells. Furthermore, the combination of Trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in OE-33 cells, which is positive for both HER2 and MET, and resistant to trastuzumab alone. Conclusion: Collectively, these data support that targeting MET with Savolitinib and capmatinib efficiently suppresses the growth of MET amplified GC cells. Moreover, MET inhibitor could synergize with trastuzumab to induce the therapeutic effect on the HER2 and MET amplified GC cells. Further studies are warranted to test this concept in clinical setting. Citation Format: Jin-Soo Kim, Sungyoul Hong, Mi Young Kim. Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal 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 6376.

Abstract 160: IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth

Abstract Lung cancer is the leading cause of cancer-related death in the U.S. and worldwide. Non-small cell lung cancer (NSCLC) represents 80-85% of all lung cancer diagnoses, most of which present as advanced disease with poor prognoses. Despite existing treatment options, the overall 5-year survival rate of lung cancer patients is about 17%. Many patients are diagnosed with advanced stage disease that frequently progresses after developing resistance to therapy. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies to improve the prognoses of patients. We recently demonstrated that dopamine and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP, promote NSCLC growth using functional studies, orthotopic mouse models, and patient-derived specimens. Specifically, we previously reported that DARPP-32 isoforms promote NSCLC cell migration through activation of non-canonical NF-κB2 signaling by establishing a direct physical interaction with inhibitory kappa B kinase-alpha (IKKα). Therefore, we first sought to determine whether IKKα directly phosphorylates DARPP-32. Indeed, we observed that kinase active IKKα protein phosphorylates DARPP-32 protein at the Thr-34 position using in vitro kinase assays. Given that phosphorylated DARPP-32 acts as a potent inhibitor of protein phosphatase 1 (PP1), we assessed PP1 activity upon transient overexpression of IKKα in DARPP-32-overexpressed NSCLC cells. Our findings suggest phosphorylation of DARPP-32 by IKKα results in decreased PP1-mediated phosphatase activity. This DARPP-32-mediated inhibition of PP1 was not observed in NSCLC cells stably overexpressing a form of DARPP-32 that cannot phosphorylated at the Thr-34 residue (via T34A mutation), suggesting IKKα protein phosphorylates DARPP-32 at the Thr-34 position to stimulate DARPP-32-mediated inhibition of PP1 activity. Correspondingly, we have observed increased phosphorylation of ERK protein, a kinase regulated by PP1, upon pharmacological inhibition of PP1 as well as following overexpression of IKKα protein. Ablation of IKKα via lentiviral transduction reduces NSCLC cell growth in soft-agar colony formation assays. Furthermore, using an orthotopic human NSCLC cell line-derived xenograft mouse model, we have shown that depletion of IKKα reduces tumor growth. Taken together, our findings suggest that IKKα contributes to NSCLC growth by interacting with DARPP-32 to stimulate oncogenic kinase activity through the inhibition of PP1 phosphatase activity. Citation Format: Sk. Kayum Alam, Li Wang, Zhu Zhu, Luke H. Hoeppner. IKKα interacts with DARPP-32 to promote lung adenocarcinoma growth [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 160.

Abstract 5951: Synergistic efficacy of sulforaphane and progesterone against endometrial cancer

Abstract Introduction: Worldwide, endometrial cancer (EC) is the most common gynecological malignancy with increasing rate of incidence and mortality. While progesterone-based hormone therapy is a well-accepted conservative therapeutic option for EC patients, loss of progesterone receptor (PR) in long-term treatment leading to hormonal resistance and disease recurrence is still an area of concern. We demonstrated potent anti-cancerous activity of sulforaphane against EC in vitro and in vivo and hypothesized that it will activate PR and overcome progesterone therapy limitations. The purpose of this study was to evaluate the interaction of sulforaphane and progesterone against EC. Methods: The in vitro synergistic interaction between sulforaphane and progesterone was investigated with isobologram analysis of plotting MTT metabolic viability assay results of EC cells treated with drugs individually and in combination using CompuSyn software. ATP, soft-agar colony formation and invasion assays were performed to evaluate the combined drug effects on cell viability, anchorage independent colony formation and invasion properties, respectively. Mitochondrial membrane potential and caspase-3 activity were measured by JC-1 and caspase-3 detection ELISA kit, respectively. Protein expression was accessed by western blot. Progesterone resistant EC cells were developed by treating cells with 30µM of progesterone for more than 3 months. Results: Sulforaphane demonstrated strong synergy or additive effects with progesterone in the EC cell lines tested. Sulforaphane in combination with progesterone significantly decreased levels of cellular ATP, anchorage-independent colony formation, invasion and mitochondrial membrane potential. Furthermore, combination of sulforaphane and progesterone increased caspase-3 activity as compared to individual drug or controls, suggesting induction of apoptosis. Western blot results demonstrated an increased expression of cleaved PARP, with reduced expression of total caspase-3 and PCNA in the combination groups as compared to control and individual drugs. Sulforaphane alone or in combination showed increased expression of PR-A while it had minimal effects on PR-B. Further, in all EC cell lines, the combination of sulforaphane and progesterone demonstrated upregulation of PR-A/PR-B ratio. The progesterone resistant cells demonstrated less sensitivity to progesterone as compared to parent cell lines, however it was found to be sensitive to sulforaphane. Conclusions: Sulforaphane demonstrated synergistic activity with progesterone leading to increased apoptosis and reduced proliferation. Furthermore, sulforaphane exhibited potent toxicity to progesterone resistance cells. Sulforaphane in combination with progesterone could overcome progesterone therapy limitation by re-activation of PR. Funding: Administrative Suppl (P30CA225520) and PHF (GRF00005456) Citation Format: Rajani Rai, Swati Choudhary, Doris M. Benbrook, Vishal Chandra. Synergistic efficacy of sulforaphane and progesterone against endometrial 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 5951.

Abstract 357: Characterization of KRas pathway inhibitors in 2D and 3D screening formats

Abstract The KRas/Raf/MEK signalling axis has been identified to play a critical role in the formation of cancer, resulting in several investigational new drugs targeting this pathway. Identification of appropriate drugs is hampered, however, by the assumption that a three-dimensional setting may be required to observe significant inhibitory effects, possibly due to alternate gene expression and protein activity in such a “closer-to-physiology” setting. In order to explore that topic, in the current study we have made use of our 140 cell lines (CL)-2D proliferation panel (3 days incubation) and our 100CL-3D soft agar panel (&amp;gt; 7 days incubation). On these platforms, we have compared inhibitors targeting the KRas/Raf/MEK pathway via either KRas:SOS interaction (e.g. BI3406), the KRas mutation at G12C (e.g. AMG510), or Raf-kinase (e.g. AZ628) and MEK1/2-kinase activity (e.g. AZD6244). It showed that especially the Raf- and MEK kinase inhibitors exhibited significantly enhanced potency in the 3D soft agar setting in the majority of the cell lines tested. The other inhibitors also showed an increased potency under soft agar conditions, however, on clearly fewer cell lines and usually not as strongly. For all kinds of inhibitors tested, the most significant changes in potency were associated with enhanced efficacy as observed by stronger cell death induction. Differences may be attributable to three-dimensional growth, but also to the elongated duration of the soft agar assay. For selected conditions, we addressed this topic by analyzing the inhibitor impact on 3D spheroid growth after shorter incubation times. Our results show that 3D growth analysis either in the soft agar or spheroid setting clearly supports the development of KRas/Raf/MEK pathway inhibitors. Citation Format: Katharina Schaich, Daniel Feger, Oliver Siedentopf, Sarah Ulrich, Jan Erik Ehlert. Characterization of KRas pathway inhibitors in 2D and 3D screening formats [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 357.

Abstract 1616: Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs

Abstract Patients with hereditary retinoblastoma (RB), an inherited autosomal dominant cancer disordercaused by germline mutations/deletions in the RB1 gene, have a &amp;gt;400 fold increased incidenceof osteosarcoma (OS), suggesting a strong mechanistic link between RB1 loss andosteosarcomagenesis. Although mice offer many advantages when conducting cancer research,unlike humans, Rb1 knockout mice do not develop OS, suggesting the urgent requirement foralternative disease models to understand how RB1 mutation leads to osteosarcomagenesis.Here, we generated patient-derived induced pluripotent stem cells (iPSCs) by reprogramming RBand healthy control fibroblasts and then created isogenic controls by correcting RB1 mutationusing CRISPR/Cas9. Performing in vitro soft-agar assay and in vivo xenografts, we foundpotential tumorigenic ability in RB osteoblasts (OBs) but not control OBs. In order to gain insightsinto RB1 loss associated osteosarcomagenesis, we compared global transcripts among RB andcontrol OBs. GSEA analysis indicated that OS associated genes are specifically enriched in RBOBs in comparison with control OBs, demonstrating that RB OBs acquire OS characteristics inthe absence of additional gene alterations. GO analysis revealed that genes involved in the mitoticcell cycle are enriched in RB OBs. Although RB1 is known to controlling G1/S related geneexpression by inhibiting E2F transcription factors, it is still nebulous if RB1 plays a role inregulating mitotic genes.To comprehensively identify preferential binding of RB1 to specific promoters in OBs, weconducted RB1 ChIP-seq to map genome-wide RB1-binding sites in iPSC-derived OBs. SinceE2F3a, a RB1 repressed transcription factor, is enriched expressed in OBs, we also performedE2F3a ChIP-seq to define whether these RB1-repressed targets are E2F3a-activated targets.Using the nonbiased de novo motif search algorithm DME, we further identified the most highlyenriched RB1-binding motif in all conditions and cross-compare with the E2F3a motifs. Weconfirmed that RB1 and E2F3a co-occupy the mitotic regulator promoters by ChIP-PCR. IHCstudies of clinical OS specimens supported the clinical correlation between RB1/E2F3a andmitotic regulators. High mitotic regulator genes expression is correlated with poor prognosis. Ourresults demonstrated that the mitotic regulators are co-regulated by both RB1 and E2F3a andthey play roles in osteosarcomagenesis.Taken together, our findings demonstrated multiple OS-related phenotypes in human RB iPSC-derived OBs and RB1/E2F3a-regulating mitotic regulators can be a therapeutic vulnerability inRB1-mutant OS. Citation Format: Dandan Zhu, Jian Tu, Zijun Huo, An Xu, Mo-Fan Huang, Ying Liu, Yi-Hung Chen, Ruiying Zhao, Dung-Fang Lee. Dissecting the biology and therapeutic vulnerabilities of RB1-mutant osteosarcoma using hereditary retinoblastoma iPSCs [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 1616.

Effects of five prescriptions in Synopsis of Golden Chamber on the malignant phenotype and expression of CA125, CEA, HE4 proteins in human ovarian cancer cells.

e17546 Background: Ovarian cancer(OC) is a common gynecological tumor with characteristics of low early diagnosis rate, late lack of effective treatment and high mortality. It is an important problem to find new effective treatment for ovarian cancer. Synopsis of the Golden Chamber is the miscellaneous diseases part of Treatise on Typhoid and Miscellaneous Diseases written by Zhang Zhongjing, a famous medical expert in Eastern Han Dynasty of China, and is also the earliest classical treatise on diagnosis and treatment of miscellaneous diseases, which records 205 prescriptions. This study aims to screen out formula with strong inhibitory effects on OC from 205 prescriptions of Synopsis of the Golden Chamber , and investigate the effects of screened prescriptions in Synopsis of the Golden Chamber on human ovarian cancer cell malignant phenotype and CA125, CEA and HE4 proteins expression. Methods: MTT method was applied to screen out the best effective prescription and determine the IC50 value of prescription on ovarian cancer ES-2 and OV-1063 cells. The effects of screened prescriptions on the proliferation, migration, invasion, cloning ability, cell cycle and CA125, CEA and HE4 proteins expression of ES-2 and OV-1063 cells were investigated by MTT assay, RTCA assay, soft agar assay, flow cytometry and western blot analysis. Results: Guizhi Fuling Pill, Dachaihu Decoction, Xuanfuhua Decoction, Baitouweng Decoction and Bentun Decoction were screened out, which all could obviously inhibit the proliferation of ES-2 and OV-1063 cells with dose-effect and time-dependent manner. The IC50 values of Guizhi Fuling Pill, Dachaihu Decoction, Xuanfuhua Decoction, Baitouweng Decoction and Bentun Decoction for ES-2 cell were 636.476, 315.859, 24.357, 9.519, 96.533μg/ml respectively; for OV-1063 were 403.119, 225.176, 12.977, 9.256, 115.86μg/ml respectively. The five prescriptions all significantly affected the cell morphology, proliferation, migration, invasion, cloning ability, cell cycle of ES-2 and OV-1063 cells. The five prescriptions all could decreased the protein expression levels of CA125 and CEA in ovarian cancer OV-1063 cells, while Guizhi Fuling Pill, Dachaihu Decoction, Xuanfuhua Decoction and Baitouweng Decoction decreased the expression of HE4 protein in OV-1063 cells. Conclusions: The five prescriptions in Synopsis of the Golden Chamber can inhibit the malignant phenotype of ovarian cancer, which may be related to the inhibition of CA125, CEA and HE4 proteins expression.

ATF4/TXNIP/REDD1/mTOR signaling mediates the antitumor activities of liver X receptor in pancreatic cancers.

Limited by difficulties in early detection and availabilities of effective treatments, pancreatic cancer is a highly malignant disease with poor prognosis. Nuclear receptors are a family of ligand-dependent transcription factors that are highly druggable therapeutic targets playing critical roles in human physiological and pathological development, including cancer. In this study, we explored the therapeutic potential as well as the molecular mechanisms of liver X receptor (LXR) agonist GW3965 in pancreatic cancer. Soft-agar colony formation assay, xenograft tumors, Oligonucleotide microarray, Reverse transcription real-time polymerase chain reaction, Western immunoblotting and Immunohistochemistry were used in this study. We demonstrated pleotropic in vitro activities of GW3965 in pancreatic cell lines MIA PaCa-2 and BXPC3 including reduction of cell viability, inhibition of cell proliferation, stimulation of cell death, and suppression of colony formation, which translated to significant inhibition of xenograft tumor growth in vitro. By mapping the gene expression profiles, we identified the up-regulations of 188 and the down-regulations of 92 genes common to both cell lines following GW3965 treatment. Genes responsive to GW3965 represent a variety of biological pathways vital for multiple cellular functions. Specifically, we identified that the activating transcription factor 4/thioredoxin-interacting protein/regulated in development and DNA damage responses 1/mechanistic target of rapamycin (ATF4/TXNIP/REDD1/mTOR) signaling critically controls GW3965-mediated regulation of cell proliferation/death. The significance of the ATF4/TXNIP/REDD1/mTOR pathway was further supported by associated expressions in xenograft tumors as well as human pancreatic cancer samples. This study provides the pre-clinical evidence that LXR agonist is a promising therapy for pancreatic cancer.