Anchorage-independent Growth Research Articles

SOX1 Functions as a Tumor Suppressor by Repressing HES1 in Lung Cancer.

The development of lung cancer is a complex process that involves many genetic and epigenetic changes. Sex-determining region Y (SRY)-box (SOX) genes encode a family of proteins that are involved in the regulation of embryonic development and cell fate determination. SOX1 is hypermethylated in human cancers. However, the role of SOX1 in the development of lung cancer is unclear. We used quantitative methylation-specific polymerase chain reaction (MSP), quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, and web tools to confirm the frequent epigenetic silencing of SOX1 in lung cancer. Stable overexpression of SOX1 repressed cell proliferation, anchorage-independent growth, and invasion in vitro as well as cancer growth and metastasis in a xenograft mouse model. Knockdown of SOX1 by the withdrawal of doxycycline partly restored the malignant phenotype of inducible SOX1-expressing NSCLC cells. Next, we discovered the potential downstream pathways of SOX1 using RNA-seq analysis and identified HES1 as a direct target of SOX1 using chromatin immunoprecipitation (ChIP)-PCR. Furthermore, we performed phenotypic rescue experiments to prove that overexpression of HES1-FLAG in SOX1-expressing H1299 cells partly reversed the tumor-suppressive effect. Taken together, these data demonstrated that SOX1 acts as a tumor suppressor by directly inhibiting HES1 during the development of NSCLC.

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 5023: PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling

Abstract Background: Ovarian cancer is one of the most aggressive malignancies threatening women’s health worldwide. Its high mortality rate is largely due to symptoms not appearing until late in the disease, usually after the cancer has metastasized. Despite current state-of-the-art treatments, the prognosis for patients remains poor. New effective treatments are urgently needed to improve patient survival. High lactate production and low glucose oxidation, independent of oxygen availability, is known as the Warburg effect and is common in cancer. Phosphofructokinase 1 (PFK-1) irreversibly converts fructose 6-phosphate to fructose 1,6-bisphosphate, the first step in the Warburg effect. The platelet isoform of PFK (PFKP) regulates glycolysis and plays roles in tumorigenesis in other human cancers, the precise mechanisms has yet to be defined. Moreover, PFKP in ovarian cancer remain unknown. We hypothesize that PFKP modulates metastasis and stemness in ovarian cancer. Methods: Expression of PFKP in clinical samples, ascites-derived tumor cells, normal ovarian epithelial cells (HOSEs) and ovarian cancer cell lines was determined by immunohistochemistry/immunofluorescence and qPCR/immunoblotting, respectively. The correlation between PFKP and clinicopathological parameters was analyzed. Ectopic expression or knockdown of PFKP followed by migration, invasion, sphere formation and soft agar assays were performed in ascites-derived tumor cells and ovarian cancer cell lines to investigate the effects on metastasis, stemness and malignant transformation respectively. Additionally, the related signaling pathway was explored by qPCR and immuoblotting. Results: Up-regulation of PFKP in clinical samples, ascites-derived tumor cells and ovarian cancer cell lines was found when compared to HOSEs. Higher PFKP was correlated to shorter overall and disease-free survival. Functionally, knockdown of PFKP by siRNA/shRNA approaches in ascites-derived tumor cells, ES-2 and HeyA8 cells decreased migration, invasion, sphere formation and colony formation in soft agar, which was accompanied by decreased expression α5 integrin, pERK and MMP9. Ectopic expression of PFKP in SKOV-3 cells showed opposite effects. Intriguingly, we found that MEK-1 inhibitor (U0126) or an anti-MMP9 neutralizing antibody could block PFKP-mediated migration/sphere formation abilities. Conclusion: PFKP was overexpressed in ovarian cancer and regulates metastasis, stemness and anchorage-independent growth. α5 integrin and ERK/MMP9 signaling were involved in such functional effects. PFKP could be a potential prognostic marker and therapeutic target for ovarian cancer. Citation Format: Ruiqian Zhang, Michelle K.Y. Siu, Xuetang Mo, Mingo M.H. Yung, Jingjing Wang, Yuxin Jiang, Annie N.Y. Cheung, Hextan Y.S. Ngan, Karen K.L. Chan. PFKP contributes to ovarian cancer metastasis, stemness and anchorage-independent growth via α5 integrin and ERK/MMP9 signaling. [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 5023.

Abstract 3858: Exploring Exportin-1 as a therapeutic vulnerability in squamous cell carcinoma

Abstract Introduction: Squamous cell carcinomas (LUSCs) account for 25-30% cases of non-small cell lung cancers, making them the second most common histology of lung cancer after adenocarcinomas. For the latter, molecular characterization has defined tumor subsets with discrete driver alterations sensitive to targeted inhibitors, leading to significant improvement in patient survival. However, for LUSCs, to date no specific drivers have been described that are amenable for pharmacological targeting and no targeted therapies are approved for use in this setting. Despite decades of research, LUSCs are still treated with cytotoxic chemotherapy, and now concomitant immunotherapy, which does not achieve durable responses in most patients. Thus, identification of targetable vulnerabilities in this setting remains a critical unmet clinical need. Preliminary data from our lab has identified the nuclear exporter exportin 1 as a therapeutic vulnerability in small cell lung carcinomas. Exportin 1 (XPO1) inhibition with Selinexor, a drug approved for clinical use in the setting of hematological malignancies, induces significant sensitivity in combination with cisplatin and irinotecan. Here, we explored the role of XPO1 as a therapeutic target in LUSC. Methods: We performed a comprehensive multi-omic molecular characterization of a library of LUSC patient-derived xenografts (PDXs) (N=28, with 17 clinical samples being matched pairs). IHC, RNA-seq and NGS via MSK-IMPACT were performed on 27 samples. To examine the potential of XPO1 inhibition as sensitizer to chemotherapy in LUSC, we performed genetic and pharmacological inhibition experiments in LUSC cell lines exhibiting high XPO1 expression. Results: Of the 28 models, 19 samples (68%) were from primary tumors and 9 samples (32%) from metastasis. The sample set were roughly split in half with regard to treatment status; 15 samples (54%) were treatment naïve and 13 sample were treated (46%).XPO1 is highly expressed in LUSCs clinical specimens compared to other tumor types, and its knockdown reduces tumorigenic features of LUSC cell lines with high XPO1 expression, including proliferation and anchorage-independent growth. Targeted XPO1 inhibition with selinexor induces chemotherapy sensitization to carboplatin and paclitaxel, drugs currently used in the treatment of LUSC, as depicted by high cytotoxic synergy scores of selinexor with either drug. Conclusion: Our data suggest that XPO1 expression may exert pro-oncogenic effects in LUSC, consistent with its upregulation in this tumor setting, and that its inhibition with selinexor may strongly sensitize to chemotherapy. Assessment of efficacy of combination therapies with selinexor in vivo will assess the potential of these combinations as a therapeutic approach for LUSC tumors. The clinical availability of selinexor would allow immediate clinical translation of the results generated in this project. Citation Format: Vidushi Durani, Rebecca Caeser, Charles M. Rudin, Alvaro Quintanal-Villalonga, Harsha Sridhar, Parvathy Manoj, Sam E. Tischfield, Marina Asher, Umeshkumar Bhanot, Jacklynn V. Egger, Nicholas D. Socci D. Socci, Nisargbhai S S. Shah, Elisa de Stanchina, Natasha Rekhtman. Exploring Exportin-1 as a therapeutic vulnerability in squamous cell carcinoma. [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 3858.

Abstract 12: Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression

Abstract KEAP1 negatively regulates the transcription factor NRF2, which regulates the response to oxidative stress. Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases, leading to constitutive activation of NRF2 and resistance to therapy in patients. To study the role of NRF2 activation in lung cancer, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and performed a thorough interrogation of how these mutations affect tumor initiation and progression. Following targeted activation of these alleles in the lung with adenoviral-Cre, immunohistochemical analysis of Nrf2 and its target Nqo1 revealed that the Keap1R554Q/R554Q mutant was the most activating towards Nrf2, followed by the Nrf2D29H/+ and Keap1R554Q/+ mutants. We found that chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. We also found that constitutive Nrf2 activation promotes lung tumor initiation and early progression of KrasG12D cells to low-grade tumors. To analyze the influence of Keap1/Nrf2 mutation on later progression, we crossed these mutations into the KrasG12D; Trp53fl/fl model of lung adenocarcinoma. Surprisingly, we found that Keap1R554Q/R554Q mutation blocked progression to advanced-grade tumors. We also found that advanced-grade tumors downregulated Nrf2 expression and activity, suggesting that excess Nrf2 activation is detrimental to tumor progression. Next, to test whether the progression block in the Keap1R554Q/R554Q model was dependent on Nrf2, we performed single copy deletion of Nrf2. We found that lowering Nrf2 levels in this model rescued the impairment in tumor progression. Finally, using a NSCLC cell culture system of NRF2 overexpression, we found that high NRF2 levels impaired cell proliferation, viability, and anchorage-independent growth. In our in vitro studies, we observed that NRF2 expression was rapidly downregulated to basal levels to restore proliferation. Our findings indicate that NRF2 promotes lung tumor initiation and early progression but impairs late progression in a dosage-dependent manner. Our preliminary data also suggests that NRF2 hyperactivation is detrimental to tumor cell proliferation. Collectively, our results establish the context-dependence and threshold for NRF2 during the tumorigenic process. Future work will include interrogating the mechanism of NRF2 downregulation in vivo and use of additional cell lines and systems to investigate how NRF2 overexpression can impair tumor cell proliferation. Citation Format: Janine M. DeBlasi, Aimee Falzone, Samantha Caldwell, Yun Pyo Kang, Nicolas Prieto-Farigua, Justin R. Prigge, Edward E. Schmidt, Iok In Christine Chio, Florian A. Karreth, Gina M. DeNicola. Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression [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 12.

Abstract 1397: Collagen COL26A1 promotes papillary thyroid cancer invasion and metastasis and correlates with poor survival and clinical outcomes

Abstract Papillary thyroid cancer (PTC) is the most common cancer in women under 25 years of age and is increasing in incidence. While PTC is often slow-growing and surgically resectable, recurrence years to decades after initial surgery occurs in ~20% of patients (now only in their 40s/50s), with mortality of 38-69%. The identification of both prognostic biomarkers and actionable therapeutic targets for high-risk PTC at diagnosis is critically important and an unmet need. RNASeq analysis of 45 PTC and matched normal patient tissues from our biobank identified collagen 26A1 (COL26A1) as significantly upregulated in patients with higher risk extrathyroidal extension, lymph node metastasis, and multifocal tumors, compared to those with localized disease. Kaplan Meier survival curves of The Cancer Genome Atlas (TCGA) data confirmed that increased COL26A1 expression significantly decreases survival probability by 25% (p=0.0086). Further analyses of TCGA data indicated that COL26A1 expression significantly correlated with clinical attributes including MACIS score (q=0.001), differentiation score (q=0.025), and tumor stage (q=0.025). Data from our in vitro culture PTC model system indicated that COL26A1 expression was increased 3X in PTC cell line K1, compared to immortalized “normal” thyroid epithelial cells, NThy-ori-3-1. Two CRISPR guides designed to target COL26A1 introduced into K1 resulted in repression of COL26A1 RNA and protein levels by 50% and 70%, respectively. COL26A1 repression decreased critical tumor phenotypes of aggressiveness and metastasis including proliferation (30%), clonogenicity (33%), anchorage-independent growth (37%), cell motility (43%), invasion (30%) and migration (33%). Cell-to-cell adhesion and cell-matrix adhesion also decreased (36% and 25%, respectively). Thus, COL26A1 may have use as a prognostic marker and actionable target for small molecule inhibitors in PTC. Citation Format: Michelle Carnazza, Danielle Quaranto, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Collagen COL26A1 promotes papillary thyroid cancer invasion and metastasis and correlates with poor survival and clinical outcomes [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 1397.

Abstract 324: Nickel exposure alters extracellular vesicles biogenesis to carry NUPR1 and mediate cancer progression

Abstract Nickel (Ni), a group I human carcinogen by the International Agency for Research on Cancer (IARC) alters expression of various oncogenic molecules in cells resulting in adoption of cancer cell phenotypes, such as anchorage-independent growth, migration, and invasion. Cancer cell phenotypes are altered by internal genetic or epigenetic changes or in response to external signals mediated by soluble proteins or extracellular vesicles. However, few studies have linked extracellular vesicles (EVs) in response to environmental pollutants, such as Ni. Nuclear Protein 1 (NUPR1) is a stress response protein that is upregulated in cancer cells and it is also upregulated in cells following chronic Ni exposure. However, the role of NUPR1 in EV biogenesis or EV-mediated cancer progression is unknown. To explore the potential role of EVs in Ni-induced cancer progression, this study examined the effect of Ni-exposure on EV signature. Sizes and concentration of EVs from BEAS-2B cells, 6 weeks-Ni-treated BEAS-2B cells (Ni-6W) and Ni-transformed BEAS-2B cells (Ni-3) were identified by nanoparticle-tracking analysis (NTA) and transmission electron microscopy (TEM). In the results, showed that Ni-6W and Ni-3 released more EVs compared to the control. The transcription expression levels of EVs biogenesis markers, CD63, CD82 and Flotillin-1 (FLOT) were coincidentally increased in Ni-6W and Ni-3 compared to the BEAS-2B. We further identified that inhibition of NUPR1 by using the shRNA or NUPR1 inhibitor ZZW-130 can rescue the increased releasing of EVs induced by Ni exposure. Here we demonstrated that Ni exposure elevated EV release and that these Ni-altered EVs contributed to a pro-inflammatory environment associated with tumorigenesis and cancer progression. Moreover, we provided a new method of regulating EVs biogenesis by targeting NUPR1, this would be crucial for EV-based cancer therapy. Citation Format: Shan Liu, Angelica Ortiz, Juan Iovanna, Max Costa. Nickel exposure alters extracellular vesicles biogenesis to carry NUPR1 and mediate cancer progression [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 324.

Abstract 330: EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling

Abstract Bladder cancer (BLCA) is the sixth most common cancer in the U.S with 83,730 new cases, accounting for 17,200 deaths in the year 2021 and 212,536 deaths worldwide. EP300, a histone acetyltransferase gene, is mutated in ~17% of BLCA and in other cancer types such as endometrial cancer. The mechanisms by which EP300 mutations contribute to tumorigenicity of BLCA is not currently understood. To determine the phenotypic effects of loss-of-function EP300 mutations, we generated isogenic BLCA cells with EP300 knocked out (KO) using CRISPR/CAS9. Conspicuous changes in phenotypes consistent with more aggressive forms of cancer were observed in EP300-null BLCA clones, including luminal-basal histotype plasticity, anchorage-independent growth, enhanced in vitro and in vivo cell proliferation and enhanced invasion in Boyden chamber assays. To identify mechanisms whereby EP300 KO promotes increased oncogenicity, we performed phospho Tandem Tag Mass Spectrometry (TMT MS) using lysates from parental and EP300 KO cells. A notable finding was significantly increased expression of phosphorylated STAT3 (pSTAT3), which we subsequently confirmed by western blot. siRNA knockdown and selective inhibitor experiments indicated JAK1 as the upstream activator of pSTAT3 in BLCA cells. Studies of conditioned media (CM) from the EP300 KO cells using ELISA and neutralizing antibodies demonstrated that secreted IL-6 ligand and soluble IL-6R in the CM drove STAT3 activation through the IL-6 family common signaling subunit, gp130, i.e. trans-signaling. BLCA RT112 cells express an oncogenic FGFR3-TACC3 fusion and are sensitive to the FDA-approved FGFR kinase inhibitor erdafitinib. pSTAT3 expression was not dependent on FGFR3 signaling in EP300 KO RT112 cells, and EP300 KO was sufficient to confer erdafitinib resistance in a BLCA context. In sum, our results uncover that EP300 loss enhances IL-6/JAK/STAT3 signaling which promotes BLCA tumorigenesis and FGFR inhibitor resistance. Our findings elucidate a role for cytokine induced sterile inflammation in EP300-mediated tumorigenesis in bladder cancers and suggest that targeting the IL-6/JAK/STAT3 axis may represent a novel therapy strategy to overcome FGFR3 inhibitor resistance. Citation Format: Sizhi P. Gao, James A. Rodrigues, Amanda R. Sabel, Jiaqian Luo, Chen Ziyu, Xinran Tang, Eduardo A. Mascareno, Naryan Rustgi, Hikmat Al-Ahmadie, Kwanghee Kim, Eugene J. Pietzak, Gopakumar V. Iyer, David B. Solit. EP300 loss drives tumorigenesis in bladder cancer via activation of IL-6/JAK/STAT3 signaling [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 330.

Abstract 300: Mechanisms of growth hormone action in the aging tissue microenvironment

Abstract The aging tissue microenvironment undergoes progressive changes enabling age-related pathologies. Senescent cells increase with age, and the secretome released from senescence cells (SASP) affects neighboring cells, often promoting proliferation, tissue inflammation, angiogenesis, and, depending on tissue type, creating a milieu favoring neoplasia. We showed that local non-pituitary growth hormone (GH) accumulates with age in human colon tissue, is induced in response to DNA damage, and suppresses DNA repair. We now show that GH is a SASP component. To examine SASP-derived GH actions on the tissue microenvironment, we co-cultured intact human 3D intestinal organoids with organoids infected with lentivirus expressing either hGH (lentiGH) or vector (lentiV) for 5 weeks. Using whole-exome sequencing to evaluate somatic mutations and somatic copy number alterations, we found that local paracrine GH emanating from lentiGH organoids increases chromosomal instability in intact organoid cells, inducing somatic mutations including deletions, breakends, duplications, and insertions, as compared to organoids co-cultured with lentiV organoids. Mechanisms for chromosomal instability observed in these organoids likely occur as a consequence of GH-induced suppression of DNA damage repair proteins including pATM, pATR, and pBRCA2, leading to DNA damage accumulation, and, in the long run, favoring cell transformation. KEGG pathway analysis of intact organoids subjected to paracrine GH exposure identified changes in focal adhesion pathways (p=0.0016), consistent with observed increased migration, invasion, and anchorage-independent growth of normal human colon cells (hNCC) exposed to GH. Furthermore, IPA analysis revealed EMT pathway changes (p=0.0018),,also confirmed by showing increased EMT transcription factors Twist2 and Snai1 expression.Although senescence has been defined as irreversible proliferative arrest, recent studies now show that senescence can be reversed. Treating hNCC with etoposide to induce senescence and culturing senescent cells in the presence of GH for 10 days resulted in p53 suppression, enabling cells to re-enter the cell cycle, as evidenced by induced Ki67, a marker of proliferation. These post-senescent cells also acquired the stem cell marker SOX2 and exhibited increased Twist2, consistent with anchorage-independent growth and increased migration as compared to senescence cells not treated with GH. These results identify local paracrine GH as an important determinant of the local tissue microenvironment promoting age-associated changes consistent with a pro-neoplastic milieu. Citation Format: Vera Chesnokova, Svetlana Zonis, Tugce Apaydin, Christian Wang, Robert Barrett, Shlomo Melmed. Mechanisms of growth hormone action in the aging tissue microenvironment [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 300.

Abstract 3753: miR-567 modulates the progression of melanoma cells and functions of macrophages

Abstract Purpose: Target therapy and immunotherapy are advancements in the treatment of melanoma; unfortunately, a subset of patients does not benefit or produces drug resistance. Therefore, an alternative strategy should be investigated to further improve clinical benefits. Macrophages are the abundant immune cells related to the progression of melanoma and resistance to drugs. In this study, we explored the novel roles of miR-567 in the progression of melanoma and effects of macrophages. Methods: miR-567 expression in nevus and melanoma tissues was analyzed by in situ hybridization. miR-567 was transfected into melanoma cells to investigate its biological effects through functional assays such as proliferation, colony formation, soft agar, and migration assays. In addition, miR-567-related signal transduction pathways in the melanoma cells were studied by western blot and next-generation sequencing analyses. Different phenotypes of macrophages polarized from THP-1 monocyte cells were used as in vitro model to investigate the effects of miR-567 on macrophages. Results: Our results showed that miR-567 expression levels were decreased in melanoma cells compared to melanocyte cells. Consistently, expression of miR-567 was 0.403-fold lower in melanoma tissues (n=118) as compared to nevus tissues (n=40). In addition, the receiver operating characteristic presented that the area under the curve value of miR-567 is 0.9495. Importantly, higher expression of miR-567 enhanced the overall survival of melanoma patients. Overexpression of miR-567 significantly reduced proliferation, survival, anchorage-independent growth, migratory, and invasive abilities of melanoma cells and BRAF-inhibitor resistant cells. Furthermore, our results demonstrated that IGF1R, E2F1, and Cyclin B2 are direct targets of miR-567 and knockdown of these genes attenuated proliferation and survival of melanoma cells. Interestingly, introduction of miR-567 downregulated MAPK/ERK and PI3K/AKT pathways in melanoma cells and regulated multiple pathways related to immune response. Of note, overexpression of miR-567 reduced the promoting melanoma growth induced by M2 or melanoma-associated macrophages, indicating the involvement of miR-567 in regulation of macrophages. Conclusion: miR-567 could be served not only as biomarkers but also as potential molecular target for prevention of melanoma progression. Citation Format: Mai-Huong Thi Nguyen, Chen-Huan Lin, Yu-Chi Huang, Mu-Shiun Tsai, Ming-Hong Chen, Azusa Miyashita, Satoshi Fukushima, Nianhan Ma. miR-567 modulates the progression of melanoma cells and functions of macrophages. [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 3753.

Abstract 3564: SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors

Abstract Background: MPNSTs are highly aggressive soft tissue sarcomas. Recurrent genetic mutations have been linked to its tumorigenesis, including the loss of functional polycomb repressive complex 2 (PRC2). This results in an altered transcription landscape characterized by the global loss of methylation on histone H3 lysine 27 (H3K27me3) and increased super-enhancer (SE) -associated histone acetylation. We have previously identified SNAI2 as a core SE-driven transcription factor (TF) that is essential for the growth of MPNST. Although SNAI2 is known to be critical for oncogenic processes, including epithelial-to-mesenchymal transition (EMT) and dedifferentiation, its role in maintaining MPNST survival through transcriptomic and epigenetic regulation has not been explored. Methods: Using CRISPR mediated knockout (KO), we targeted the SNAI2 DNA binding domain and confirmed KO via western blot, followed by in vitro phenotypic characterization, including 2D growth assays, anchorage-independent colony formation assays, and scratch assays. Additionally, an integrative analysis of SNAI2 genomic distribution and the transcriptomic effect of its KO was performed using chromatin immunoprecipitation coupled with DNA sequencing (ChIPseq) and RNA sequencing (RNAseq). Finally, an inducible re-expression system was employed to examine the effects on the chromatin landscape with SNAI2 rescue. Results: Genetic loss of SNAI2 slowed the growth of MPNST cells by 50% in comparison to the control. In vitro tumorigenic assays revealed that SNAI2 KO diminished the cells’ capacity in invasion but not in anchorage-independent cell growth. Analysis of the RNAseq results revealed that SNAI2 loss upregulated 790 genes and downregulated 1623 genes (log2fold change> 1, p<0.01), indicating a dominant transcriptional activation role. Gene Set Enrichment Analysis of these SNAI2-regulated genes revealed upregulation of hallmark Myc targets (normalized enrichment score (NES)=2.6, FDR=0.0), whilst genes involved in response to alpha interferon proteins were downregulated (NES=-1.87, FDR=0.002). Interestingly, genes defining EMT were upregulated (NES=1.6, FDR=0.006) in cells upon SNAI2 loss, contrasting its known repressive role in this biological process in other systems. Through integrative analysis of the RNAseq and ChIPseq data, we demonstrate SNAI2 binding to promoters of its regulated targets. Downstream targets of interest include CRELD1, a gene involved in cell adhesion, where SNAI2 KO caused loss of promoter binding and gene downregulation (p<0.001). Conclusion: SNAI2 is an essential SE-driven TF in MPNST cells. Transcriptomic and genomic profiling of SNAI2 through loss- and gain-of-function experiments revealed that it is a critical TF that is involved in key biological processes. Future efforts will focus on designing methods to employ targeted degradation of this essential TF and test preclinical efficacy in treating MPNST. Citation Format: Hilda Jafarah, Béga Murray, Jack Shern, Xiyuan Zhang. SNAI2 enhances oncogenesis in malignant peripheral nerve sheath tumors. [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 3564.

Abstract 6734: Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor that occurs in childhood and the most common cancer in infancy. Fewer than 5% of neuroblastomas occur in adolescents and young adults (AYAs), for whom the disease has an indolent and fatal course. Chen et al. previously reported that missense mutations in the protein- tyrosine kinase domain of anaplastic lymphoma kinase (ALK) occur in approximately 10% of cases of sporadic neuroblastoma, and these missense mutations result in the activation of the ALK protein kinase domain, which plays a key role in the tumorigenic process as seen in ALK fusion proteins. Since the initial discovery of the NPM-ALK fusion protein in human anaplastic lymphoma cell lines, more than 24 different ALK fusion proteins have been discovered in various malignancies. Till date, ALK-containing chimeric proteins have not been reported in neuroblastoma. In this study, a translocation within chromosome 2p and 4q was found to bring about the formation of an in-frame fusion gene that was composed of portions of the teneurin transmembrane protein 3 (TENM3) gene and ALK gene in tumor cells from an AYA patient with neuroblastoma. The patient was 19-year-old female diagnosed with stage 4 neuroblastoma (unfavorable histology, poorly differentiated subtype, MYCN DNA not amplified). Computed tomography revealed a mass in the left adrenal gland with bone, spine, liver, and lung metastasis. ALK rearrangement was confirmed via FISH analysis. Crizotinib resulted in no disease progression for 5 months, except for metastatic spinal disease, but it did not exhibit high efficacy compared with its effects against lymphoma and lung cancer. This 5′ in-frame fusion partner gene in neuroblastoma has not yet been identified, and its role is not well-known in oncogenesis. We generated the cell line with the expression of the full length TENM3-ALK cDNA in NIH-3T3 cells led to the formation of a fusion protein. A cell-based evaluation of this predicted ALK protein variant revealed activation of downstream targets-STAT3, AKT and ERK- and ALK inhibitors, crizotinib or lorlatinib, treatment inhibited the growth of xenograft tumors with stable TENM3-ALK expression. We determined whether TENM3-ALK affects transformation and proliferation using a soft agar colony formation assay. As expected, the cells that expressed an empty vector and wild type ALK line did not produce a considerable number of colonies, whereas TENM3-ALK transfected cells showed anchorage-independent growth, resulting in the production of a higher number of colonies. The evidence for the role of ALK receptor signaling in stimulating neuroblastoma tumor cell growth and the demonstrated in vitro and in vivo efficacy of the ALK selective inhibitors in this study provide biological and clinical justification for the further exploration of this combination and testing in patients with recurrent or refractory neuroblastoma. Citation Format: Mitsuteru Hiwatari, Masafumi Seki, Ryosuke Matsuno, Kenichi Yoshida, Takeshi Nagasawa, Aiko Sato-Otsubo, Shohei Yamamoto, Motohiro Kato, Kentaro Watanabe, Masahiro Sekiguchi, Satoru Miyano, Seishi Ogawa, Junko Takita. Identification of the novel TENM3-ALK fusion in an AYA case with ALK rearranged neuroblastoma [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 6734.

Abstract 6242: Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma

Abstract Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB). Transcriptional inhibitors of survivin have been tested in clinical trials. However, results have been disappointing. We thus investigated the small molecules LQZ-7I and S12 that inhibit dimerization of survivin protein and compared their growth-inhibiting efficacy with that of markedly decreased survivin expression in a novel transgenic NB mouse. Mice hemizygous for BIRC5 were crossed with TH-MYCN mice to generate BIRC5+/−/MYCNtg/+ mice. NB incidence, growth and retransplantation capacity were assessed. In vitro, LQZ-7I and S12 were used to inhibit survivin protein interactions and the effects on proliferation and cell cycle of NB cells were investigated. BIRC5 hemizygous mice developed NB although mRNA and protein expression of survivin was markedly decreased. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/−/MYCNtg/+ mice did not differ from the NB in the TH-MYCN mice, and tumor growth and survival of the mice was not affected. In stark contrast, the survivin interaction inhibitors LQZ-7I and S12 abolished anchorage-independent growth and proliferation of NB cell lines. Reduced growth of NB cells resulted from defects in mitosis induced by LQZ-7I or S12 or. LQZ-7I was the more efficient inhibitor of NB cells while S12 was not able to reduce tumor growth in a xenograft model. Taken together, these results support the notion that inhibiting survivin interaction is more efficacious in NB than inhibiting survivin transcription. Citation Format: Carmen Dorneburg, Celimene Galliger, Voker Rasche, Andre Lechel, Thomas F. Barth, Klaus-Michael Debatin, Christian Beltinger. Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma. [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 6242.

Abstract 1482: Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the highest mortalities of all malignancies, with a mere 5-year survival of ~10%. However, the current first-line treatments have poor patient outcomes, highlighting the urgent need for innovative therapeutics. The nuclear factor κB (NF-κB) is a crucial transcription factor frequently activated constitutively in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds substantial promise in PDAC diagnosis and new therapeutics. The purpose of this study is to identify novel regulation of NF-κB, with the aim of providing new diagnostic and therapeutic strategies for PDAC. Here, we report protein O-GlcNAc transferase (OGT) - mediated NF-κB activation through novel serine O-GlcNAcylation of its p65 subunit. We show that overexpression of serine-to-alanine (S-A) mutant at the O-GlcNAcylation site impaired NF-κB nuclear translocation and transcriptional activity in PDAC cells. Moreover, these S-A p65 mutants downregulate NF-κB-target genes important to major cancer hallmarks and inhibit cellular proliferation, migration, and anchorage-independent growth of PDAC cells compared to WT-p65. Interestingly, this modification happens downstream of the inhibitor of NF-κB (IκBα) degradation. Collectively, we have identified OGT-mediated serine O-GlcNAcylation of NF-κB and determined its mechanistic and cellular function in driving PDAC phenotypes. Thus, our study holds great significance as it uncovers a brand-new strategy for PDAC diagnosis and effective therapeutics development. Citation Format: Aishat Motolani, Matthew Martin, Benlian Wang, Guanglong Jiang, Yunlong Liu, Tao Lu. Critical role of OGT-mediated novel NF-κB o-glcNAcylation in pancreatic 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 1482.

The GAPDH redox switch safeguards reductive capacity and enables survival of stressed tumour cells

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response to hydrogen peroxide, leading to rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable to stimulate the oxidative pentose phosphate pathway and enhance their reductive capacity. Specifically, we find that anchorage-independent growth of cells and spheroids is limited by an elevation of endogenous peroxide levels and is largely dependent on a functional GAPDH redox switch. Likewise, tumour growth in vivo is limited by peroxide stress and suppressed when the GAPDH redox switch is disabled in tumour cells. The induction of additional intratumoural oxidative stress by chemo- or radiotherapy synergized with the deactivation of the GAPDH redox switch. Mice lacking the GAPDH redox switch exhibit altered fatty acid metabolism in kidney and heart, apparently in compensation for the lack of the redox switch. Together, our findings demonstrate the physiological and pathophysiological relevance of oxidative GAPDH inactivation in mammals.

Generation and analysis of whole-genome sequencing data in human mammary epithelial cells.

Breast cancer is the most common cancer worldwide, and advanced breast cancer with metastases is incurable mainly with currently available therapies. Therefore, it is essential to understand molecular characteristics during the progression of breast carcinogenesis. Here, we report a dataset of whole genomes from the human mammary epithelial cell system derived from a reduction mammoplasty specimen. This system comprises pre-stasis 184D cells, considered normal, and seven cell lines along cancer progression series that are immortalized or additionally acquired anchorage-independent growth. Our analysis of the whole-genome sequencing (WGS) data indicates that those seven cancer progression series cells have somatic mutations whose number ranges from 8,393 to 39,564 (with an average of 30,591) compared to 184D cells. These WGS data and our mutation analysis will provide helpful information to identify driver mutations and elucidate molecular mechanisms for breast carcinogenesis.

Isolation and Characterization of Novel Canine Osteosarcoma Cell Lines from Chemotherapy-Naïve Patients

The present study aimed to establish novel canine osteosarcoma cell lines (COS3600, COS3600B, COS4074) and characterize the recently described COS4288 cells. The established D-17 cell line served as a reference. Analyzed cell lines differed notably in their biological characteristics. Calculated doubling times were between 22 h for COS3600B and 426 h for COS4074 cells. COS3600B and COS4288 cells produced visible colonies after anchorage-independent growth in soft agar. COS4288 cells were identified as cells with the highest migratory capacity. All cells displayed the ability to invade through an artificial basement membrane matrix. Immunohistochemical analyses revealed the mesenchymal origin of all COS cell lines as well as positive staining for the osteosarcoma-relevant proteins alkaline phosphatase and karyopherin α2. Expression of p53 was confirmed in all tested cell lines. Gene expression analyses of selected genes linked to cellular immune checkpoints (CD270, CD274, CD276), kinase activity (MET, ERBB2), and metastatic potential (MMP-2, MMP-9) as well as selected long non-coding RNA (MALAT1) and microRNAs (miR-9, miR-34a, miR-93) are provided. All tested cell lines were able to grow as multicellular spheroids. In all spheroids except COS4288, calcium deposition was detected by von Kossa staining. We believe that these new cell lines serve as useful biological models for future studies.

IKKα promotes lung adenocarcinoma growth through ERK signaling activation via DARPP-32-mediated inhibition of PP1 activity

Non-small cell lung cancer (NSCLC) accounts for 80–85% cases of lung cancer cases. Diagnosis at advanced stages is common, after which therapy-refractory disease progression frequently occurs. Therefore, a better understanding of the molecular mechanisms that control NSCLC progression is necessary to develop new therapies. Overexpression of IκB kinase α (IKKα) in NSCLC correlates with poor patient survival. IKKα is an NF-κB-activating kinase that is important in cell survival and differentiation, but its regulation of oncogenic signaling is not well understood. We recently demonstrated that IKKα promotes NSCLC cell migration by physically interacting with dopamine- and cyclic AMP-regulated phosphoprotein, Mr 32000 (DARPP-32), and its truncated splice variant, t-DARPP. Here, we show that IKKα phosphorylates DARPP-32 at threonine 34, resulting in DARPP-32-mediated inhibition of protein phosphatase 1 (PP1), subsequent inhibition of PP1-mediated dephosphorylation of ERK, and activation of ERK signaling to promote lung oncogenesis. Correspondingly, IKKα ablation in human lung adenocarcinoma cells reduced their anchorage-independent growth in soft agar. Mice challenged with IKKα-ablated HCC827 cells exhibited less lung tumor growth than mice orthotopically administered control HCC827 cells. Our findings suggest that IKKα drives NSCLC growth through the activation of ERK signaling via DARPP-32-mediated inhibition of PP1 activity.

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

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

Antineoplastic Nature of WWOX in Glioblastoma Is Mainly a Consequence of Reduced Cell Viability and Invasion

Following the discovery of WWOX, research has moved in many directions, including the role of this putative tumor suppressor in the central nervous system and related diseases. The task of determining the nature of WWOX in glioblastoma (GBM) is still considered to be at the initial stage; however, the influence of this gene on the GBM malignant phenotype has already been reported. Because most of the available in vitro research does not consider several cellular GBM models or a wide range of investigated biological assays, the present study aimed to determine the main processes by which WWOX exhibits anticancer properties in GBM, while taking into account the phenotypic heterogeneity between cell lines. Ectopic WWOX overexpression was studied in T98G, DBTRG-05MG, U251MG, and U87MG cell lines that were compared with the use of assays investigating cell viability, proliferation, apoptosis, adhesion, clonogenicity, three-dimensional and anchorage-independent growth, and invasiveness. Observations presenting the antineoplastic properties of WWOX were consistent for T98G, U251MG, and U87MG. Increased proliferation and tumor growth were noted in WWOX-overexpressing DBTRG-05MG cells. A possible explanation for this, arrived at via bioinformatics tools, was linked to the TARDBP transcription factor and expression differences of USP25 and CPNE2 that regulate EGFR surface abundance. Collectively, and despite various cell line-specific circumstances, WWOX exhibits its anticancer nature mainly via a reduction of cell viability and invasiveness of glioblastoma.