Promote Tumor Initiation Research Articles

Abstract 3285: Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells

Abstract Background: STAT3 is a transcription factor that regulates various tumorigenic processes, such as tumor proliferation, metastasis, and drug resistance. Its overexpression and aberrant activation characterize many cancers (including breast, lung, ovarian, liver and colon cancer). Metastasis and drug resistance are two main causes of patient mortality. Activation of the JAK-STAT3 and the FGFR1-STAT3 pathways in breast and ovarian cancer cells promotes tumor initiation, migration, and taxol resistance. STAT3 also contributes to epithelial-mesenchymal transition and promotes 5-FU resistance in colorectal cancer cells. Its role in numerous malignancies has made STAT3 an ideal cancer therapeutic target. BP1003 is a neutral DOPC liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide targeting STAT3 mRNA. Here we determine the effects of BP1003 in combination with taxol and 5-FU on breast and ovarian cancer cells. Methods: Western blots determined the extent by which BP1003 reduced STAT3 protein levels. The effect of sequential taxol or 5-FU (5 h pre-treatment) and BP1003 treatments (96 h) on cell viability was analyzed with resazurin-based assays. Migration and colony formation assays were performed to assess the metastatic potential of treated cells. Cells pre-treated with BP1003 (96 h) and taxol (2 h) were placed in chemotaxis chambers and allowed to migrate for 5-18 h before quantification. Colony formation involved the treatment of a low number of cells with chemotherapeutic drugs for 24 h prior to the addition of BP1003 for 10-14 days. Tumor spheroids were used to mimic in vivo solid tumor growth. Three days after formation, spheroids were treated with chemotherapeutic drugs for 1 day before being treated with BP1003 for 3 additional days. Results: Treatment of BT549, SK-Br-3 and SK-Ov-3 cells with 250 μg/ml of BP1003 resulted in a 30%-50% decrease in STAT3 expression. Cell viability decreased by 20-35% with 200 μg/ml of BP1003 and by 45-60% in combination with taxol or 5-FU. BP1003 in combination with taxol also decreased the colony formation potential of BT549 cells by 20%. Migration of BT549 cells was reduced by 30% with 200 μg/ml BP1003 alone and by 50% in combination with taxol. The area of BT549 and SK-Ov-3 spheroids under BP1003 and taxol combination treatments was 40%-50% smaller than control spheroids and 20%-30% smaller than taxol mono-treatment. Conclusions: BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast and ovarian cancer cells to taxol and 5-FU. These results are in line with previous work in which BP1003 + gemcitabine displayed enhanced anti-tumor activity in pancreatic ductal adenocarcinoma. Together these results strongly suggest that BP1003 combination therapy is a novel strategy for patients with advanced solid tumors. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3285.

Tumor-Associated Inflammation: The Tumor-Promoting Immunity in the Early Stages of Tumorigenesis.

Tumorigenesis is a multistage progressive oncogenic process caused by alterations in the structure and expression level of multiple genes. Normal cells are continuously endowed with new capabilities in this evolution, leading to subsequent tumor formation. Immune cells are the most important components of inflammation, which is closely associated with tumorigenesis. There is a broad consensus in cancer research that inflammation and immune response facilitate tumor progression, infiltration, and metastasis via different mechanisms; however, their protumor effects are equally important in tumorigenesis at earlier stages. Previous studies have demonstrated that during the early stages of tumorigenesis, certain immune cells can promote the formation and proliferation of premalignant cells by inducing DNA damage and repair inhibition, releasing trophic/supporting signals, promoting immune escape, and activating inflammasomes, as well as enhance the characteristics of cancer stem cells. In this review, we focus on the potential mechanisms by which immune cells can promote tumor initiation and promotion in the early stages of tumorigenesis; furthermore, we discuss the interaction of the inflammatory environment and protumor immune cells with premalignant cells and cancer stem cells, as well as the possibility of early intervention in tumor formation by targeting these cellular mechanisms.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Regulation of Hippo signaling by metabolic pathways in cancer.

Hippo signaling is known to maintain balance between cell proliferation and apoptosis via tight regulation of factors, such as metabolic cues, cell-cell contact, and mechanical cues. Cells directly recognize glucose, lipids, and other metabolic cues and integrate multiple signaling pathways, including Hippo signaling, to adjust their proliferation and apoptosis depending on nutrient conditions. Therefore, the dysregulation of the Hippo signaling pathway can promote tumor initiation and progression. Alteration in metabolic cues is considered a major factor affecting the risk of cancer formation and progression. It has recently been shown that the dysregulation of the Hippo signaling pathway, through diverse routes activated by metabolic cues, can lead to cancer with a poor prognosis. In addition, unique crosstalk between metabolic pathways and Hippo signaling pathways can inhibit the effect of anticancer drugs and promote drug resistance. In this review, we describe an integrated perspective of the relationship between the Hippo signaling pathway and metabolic signals in the context of cancer. We also characterize the mechanisms involved in changes in metabolism that are linked to the Hippo signaling pathway in the cancer microenvironment and propose several novel targets for anticancer drug treatment.

Polymeric Hydrogels for In Vitro 3D Ovarian Cancer Modeling.

Ovarian cancer (OC) grows and interacts constantly with a complex microenvironment, in which immune cells, fibroblasts, blood vessels, signal molecules and the extracellular matrix (ECM) coexist. This heterogeneous environment provides structural and biochemical support to the surrounding cells and undergoes constant and dynamic remodeling that actively promotes tumor initiation, progression, and metastasis. Despite the fact that traditional 2D cell culture systems have led to relevant medical advances in cancer research, 3D cell culture models could open new possibilities for the development of an in vitro tumor microenvironment more closely reproducing that observed in vivo. The implementation of materials science and technology into cancer research has enabled significant progress in the study of cancer progression and drug screening, through the development of polymeric scaffold-based 3D models closely recapitulating the physiopathological features of native tumor tissue. This article provides an overview of state-of-the-art in vitro tumor models with a particular focus on 3D OC cell culture in pre-clinical studies. The most representative OC models described in the literature are presented with a focus on hydrogel-based scaffolds, which guarantee soft tissue-like physical properties as well as a suitable 3D microenvironment for cell growth. Hydrogel-forming polymers of either natural or synthetic origin investigated in this context are described by highlighting their source of extraction, physical-chemical properties, and application for 3D ovarian cancer cell culture.

NRF2: KEAPing Tumors Protected.

Alterations in the KEAP1/NRF2 pathway are found in multiple cancer types. Activation of NRF2 leads to metabolic rewiring of tumors that promote tumor initiation and progression. Here we present the known alterations that lead to NRF2 activation in cancer, the mechanisms in which NRF2 activation promotes tumors, and the therapeutic implications of NRF2 activation.

PKC is an indispensable factor in promoting environmental toxin chromium-mediated transformation and drug resistance

Hexavalent chromium [Cr(VI)] pollution is a serious environmental problem, due to not only its toxicity but also carcinogenesis. Although studies reveal several features of Cr(VI)-induced carcinogenesis, the underlying mechanisms of how Cr(VI) orchestrates multiple mitogenic pathways to promote tumor initiation and progression remain not fully understood. Src/Ras and other growth-related pathways are shown to be key players in Cr(VI)-initiated tumor prone actions. The role of protein kinase C (PKC, an important signal transducer) in Cr(VI)-mediated carcinogenesis has not been thoroughly investigated. In this study, using human bronchial/lung epithelial cells and keratinocytes, we demonstrate that PKC activity is increased by transient or chronic Cr(VI) exposure, which plays no role in the activation of Src/Ras signaling and ROS upregulation by this metal toxin. PKC in chronic Cr(VI)-treated cells stabilizes Bcl-2 to mitigate doxorubicin (an anti-cancer drug)-mediated apoptosis. After the suppression of this kinase by GO6976 (a PKC inhibitor), the cells chronically exposed to Cr(VI) partially regain the sensitivity to doxorubicin. However, when co-suppressed PKC and Ras, the chronic Cr(VI)-treated cells become fully responsive to doxorubicin and are unable to be transformed. Taken together, our study provides a new insight into the mechanisms, in which PKC is an indispensable player and cooperates with other mitogenic pathways to achieve Cr(VI)-induced carcinogenesis as well as to establish drug resistance. The data also suggest that active PKC can serve as a potential biomarker for early detection of health damages by Cr(VI) and therapeutic target for developing new treatments for diseases caused by Cr(VI).

The ion channel TRPV1 gain-of-function reprograms the immune microenvironment to facilitate colorectal tumorigenesis.

Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-permeable ion channel that acts as cellular sensor and is implicated in the tumor microenvironment cross talk. However, the functional role of TRPV1 in colorectal cancer (CRC) is still controversial. By using a TRPV1 gain-of-function model, we previously reported that hyperfunctional TRPV1 exacerbated experimental colitis by modulating mucosal immunity. Here, we found that TRPV1 gain-of-function significantly promoted tumor initiation and progression in colitis-associated cancer, as evidenced by the increase in the number and size of tumor. Systemic TRPV1 hyperactivation fostered a tumor permissive microenvironment through altering macrophage activation status and shifting the Th1/Th2 balance towards Th2 phenotype. Mechanistically, TRPV1 gain-of-function directly potentiated M1 cytokine production in macrophage and enhanced Th2 immune response by promoting Calcineurin/nuclear factor of activated T cells (NFATc2) signaling activation. In patients with CRC, TRPV1 expression was increased in tumor immune infiltrating cells. TRPV1 level was associated with CRC progression and could impact clinical outcome. Our study reveals an important role for TRPV1 in regulating the immune microenvironment during colorectal tumorigenesis. TRPV1 might be a potential target for CRC immunotherapy.

The Roles of MicroRNA-133 in Gynecological Tumors.

MicroRNAs are noncoding small RNAs that regulate gene expression posttranscriptionally. They act as a key role not only in the body development but also in many human diseases, including malignant tumors. With evidence of the complex role of miR-133 during gynecological malignancies initiation and progression are gradually emerging, miR-133 shows suppressive function by inhibiting tumor proliferation, invasion, and metastasis or acts as an oncogene by promoting tumor initiation, growth and invasion, depending on different tumor types and differentiation. In this review, we summarize the role and related regulatory methods of miR-133 in gynecological cancers. Moreover, then, we analyze and clarify the research status of other microRNAs acting on similar genes and pathways in gynecological tumors and look forward to their future research directions. This review may provide new expectations for applying miR-133 as diagnostic and prognostic biomarkers in gynecological tumors.

Circular RNA circTADA2A promotes the proliferation, invasion, and migration of non-small cell lung cancer cells via the miR-450b-3p/HMGN5 signaling pathway.

BackgroundCircular RNAs (circRNAs) have been confirmed to exert important roles in promoting tumor initiation and progression. However, the expression, effect, and underlying mechanism of circTADA2A in non-small cell lung cancer (NSCLC) remain unclear.MethodsA total of 60 paired clinical samples of NSCLC tissues and corresponding normal adjacent tissues were obtained. Quantitative real-time PCR was used to verify circTADA2A, miR-450b-3p, and HMGN5 mRNA expression. The NSCLC cell Lines A549 and H1299 were individually transfected with circTADA2A and HMGN5. The regulatory interaction between circTADA2A and miR-450b-3p was investigated by dual-luciferase reporter assay. HMGN5 protein expression was detected by Western blotting.ResultsCircTADA2A expression was significantly upregulated and correlated with poor overall survival of NSCLC patients. Functionally, circTADA2A inhibition successfully suppressed the proliferation, invasion, and migration of A549 and H1299 cells. circTADA2A functioned as a competing endogenous RNA to sponge miR-450b-3p to promote the expression of HMGN5 mRNA and protein. Furthermore, a positive relationship between circTADA2A and HMGN5 existed in NSCLC tissues. There were negative relationships between circTADA2A and miR-450b-3p as well as miR-450b-3p and HMGN5 in NSCLC tissues.ConclusionsThese findings suggest that circTADA2A might act as an oncogenic circRNA that promotes NSCLC progression by sponging miR-450b-3p and promoting HMGN5 expression, indicating that the suppression of circTADA2A could become a potential therapeutic target for restraining NSCLC.

Downregulation of PHLPP induced by endoplasmic reticulum stress promotes eIF2\u03b1 phosphorylation and chemoresistance in colon cancer

Aberrant activation of endoplasmic reticulum (ER) stress by extrinsic and intrinsic factors contributes to tumorigenesis and resistance to chemotherapies in various cancer types. Our previous studies have shown that the downregulation of PHLPP, a novel family of Ser/Thr protein phosphatases, promotes tumor initiation, and progression. Here we investigated the functional interaction between the ER stress and PHLPP expression in colon cancer. We found that induction of ER stress significantly decreased the expression of PHLPP proteins through a proteasome-dependent mechanism. Knockdown of PHLPP increased the phosphorylation of eIF2α as well as the expression of autophagy-associated genes downstream of the eIF2α/ATF4 signaling pathway. In addition, results from immunoprecipitation experiments showed that PHLPP interacted with eIF2α and this interaction was enhanced by ER stress. Functionally, knockdown of PHLPP improved cell survival under ER stress conditions, whereas overexpression of a degradation-resistant mutant PHLPP1 had the opposite effect. Taken together, our studies identified ER stress as a novel mechanism that triggers PHLPP downregulation; and PHLPP-loss promotes chemoresistance by upregulating the eIF2α/ATF4 signaling axis in colon cancer cells.

Automated Detection of Arm-Level Alterations for Individual Cancer Patients in the Clinical Setting

Copy number alterations are genetic events that promote tumor initiation and progression and are used in clinical care as diagnostic, prognostic, and predictive biomarkers. Based on the length of the alteration, they are roughly classified as focal and arm-level alterations. Although genome-wide techniques to detect arm-level alterations are gaining momentum in hospital laboratories, the high precision and novelty of these techniques pose new challenges: there is no consensus on the definition of an arm-level alteration and there is a lack of tools to compute them for individual patients. Based on 376 clinical samples analyzed with the OncoScan formalin-fixed, paraffin-embedded assay, a bimodal distribution of the percentage of bases with copy number alterations within a chromosomal arm was observed, with the second peak starting at 90% of arm length. Two approaches were tested for the definition of arm-level alterations: sum of altered segments (SoS) >90%, or the longest segment (LS) >90%. These approaches were validated against expert annotation of 25 clinical cases. The SoS method outperformed the LS method as indicated by a higher concordance (SoS, 95.2%; LS, 79.9%). Some of the discordances ultimately were attributed to human error, highlighting the advantages of automation. The increase in reliability led to the development of publicly available software and its inclusion into routine clinical practice at Geneva University Hospitals.

MiR-22 promotes stem cell traits via activating Wnt/β-catenin signaling in cutaneous squamous cell carcinoma

Emerging evidence suggests that the cancer stem cells (CSCs) are key culprits of cancer metastasis and drug resistance. Understanding mechanisms regulating the critical oncogenic pathways and CSCs function could reveal new diagnostic and therapeutic strategies. We now report that miR-22, a miRNA critical for hair follicle stem/progenitor cell differentiation, promotes tumor initiation, progression, and metastasis by maintaining Wnt/β-catenin signaling and CSCs function. Mechanistically, we find that miR-22 facilitates β-catenin stabilization through directly repressing citrullinase PAD2. Moreover, miR-22 also relieves DKK1-mediated repression of Wnt/β-catenin signaling by targeting a FosB-DKK1 transcriptional axis. miR-22 knockout mice showed attenuated Wnt/β-catenin activity and Lgr5+ CSCs penetrance, resulting in reduced occurrence, progression, and metastasis of chemically induced cutaneous squamous cell carcinoma (cSCC). Clinically, miR-22 is abundantly expressed in human cSCC. Its expression is even further elevated in the CSCs proportion, which negatively correlates with PAD2 and FosB expression. Inhibition of miR-22 markedly suppressed cSCC progression and increased chemotherapy sensitivity in vitro and in xenograft mice. Together, our results revealed a novel miR-22-WNT-CSCs regulatory mechanism in cSCC and highlight the important clinical application prospects of miR-22, a common target molecule for Wnt/β-catenin signaling and CSCs, for patient stratification and therapeutic intervention.

Abstract 1147: RNA-targeting approaches to modulate alternative RNA splicing in cancer

Abstract Alternative RNA splicing (AS) is a critical step in gene expression regulation. This differential inclusion of sequences in the mRNA is a source of functional transcriptomic diversity that allows multiple RNA isoforms to be expressed from the same gene. Dysregulation of AS is hallmark of cancer and leads to the expression of RNA isoforms that promote tumor initiation and maintenance. Understanding how tumor-associated RNA isoforms impact cancer biology is needed for the development of cancer therapies targeting AS. Here, we describe two approaches to modulate AS in cancer cells. First, we utilize CRISPR-Artificial Splicing Factors (CASFx), splicing factor domains fused to catalytically inactive Cas13 proteins, to direct splicing activity to a specific site using a guide RNA (gRNA). We demonstrate that this approach can be used to promote either exon inclusion or skipping in mRNA from several cancer-associated genes (e.g., HRAS, EP400, TRA2β, SRSF3) depending on the gRNA binding location and the splicing-factor domain fusion. Second, we develop antisense oligonucleotides (ASOs) that target a non-coding poison exon in the oncogenic splicing factor TRA2β. This exon functions as a regulatory “poison exon”, which induces RNA degradation when included, and is differentially spliced in multiple tumor types compared to their corresponding adjacent normal tissue. TRA2β-targeting ASOs promoting poison exon inclusion cause decreased TRA2β protein expression, increased cell death, and decreased proliferation across multiple cancer cell lines. Additionally, compared to cancer cells, normal cell lines were less affected by TRA2β-targeting ASO toxicity, demonstrating a therapeutic window for using this technology to specifically target cancer cells. Citation Format: Nathan K. Leclair, Laura Urbanski, Mattia Brugiolo, Marina Yurieva, Brenton R. Graveley, Albert Cheng, Olga Anczukow. RNA-targeting approaches to modulate alternative RNA splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1147.

Abstract LB244: The role of stromal Per2 in regulation of tumorigenesis

Abstract The tumor microenvironment (TME) comprised of multiple proteins, extracellular matrix(ECM) and various cell types that together promote tumor initiation, progression andhomeostasis in a cell-cell communication manner. In this study, we set to understand howthe stromal circadian clock supports the cancer cells, specifically the contribution of thecore clock gene Per2 to tumor initiation and progression. The circadian clock is anendogenous, evolutionally conserved and ubiquitously expressed pacemaker, consisting ofcell autonomous clocks and a central pacemaker located in the hypothalamussuperchiasmatic nucleus (SCN). Together these clocks synchronize numerous biologicalprocesses between the organism and its environment. Amongst these processes are DNAdamage repair, metabolism, and cell cycle. Several studies showed that disruption ofcircadian rhythms has been associated with various forms of cancer in humans and mice.While Per2 was previously suggested to be a tumor suppressor, in this study we found thatthe core clock gene Per2 in the TME is essential for tumor initiation and metastaticcolonization. Another core gene, Per1, is dispensable. We further showed that lossof Per2 in the TME leads to transcriptional rewiring at early stages of metastasesformation, and suppresses subsequent metastatic tumor progression. Thus, our resultsunravel an unexpected protumorigenic role for the core clock gene Per2 in the TME. Thesefindings may imply a non-circadian role for Per2, differentiating it from Per1 withpotential implications for therapeutic dosing strategies and treatment regimens. Citation Format: Shimrit Mayer, Lee Shaashua, Chen Lior, Hagar Lavon, Alexander Novoselsky, Ruth Scherz-Shouval. The role of stromal Per2 in regulation of tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB244.

Mitochondrial Metabolism in Carcinogenesis and Cancer Therapy.

Simple SummaryReprogramming metabolism is a hallmark of cancer. Warburg’s effect, defined as increased aerobic glycolysis at the expense of mitochondrial respiration in cancer cells, opened new avenues of research in the field of cancer. Later findings, however, have revealed that mitochondria remain functional and that they actively contribute to metabolic plasticity of cancer cells. Understanding the mechanisms by which mitochondrial metabolism controls tumor initiation and progression is necessary to better characterize the onset of carcinogenesis. These studies may ultimately lead to the design of novel anti-cancer strategies targeting mitochondrial functions.Carcinogenesis is a multi-step process that refers to transformation of a normal cell into a tumoral neoplastic cell. The mechanisms that promote tumor initiation, promotion and progression are varied, complex and remain to be understood. Studies have highlighted the involvement of oncogenic mutations, genomic instability and epigenetic alterations as well as metabolic reprogramming, in different processes of oncogenesis. However, the underlying mechanisms still have to be clarified. Mitochondria are central organelles at the crossroad of various energetic metabolisms. In addition to their pivotal roles in bioenergetic metabolism, they control redox homeostasis, biosynthesis of macromolecules and apoptotic signals, all of which are linked to carcinogenesis. In the present review, we discuss how mitochondria contribute to the initiation of carcinogenesis through gene mutations and production of oncometabolites, and how they promote tumor progression through the control of metabolic reprogramming and mitochondrial dynamics. Finally, we present mitochondrial metabolism as a promising target for the development of novel therapeutic strategies.

Inhibition of yes-associated protein suppresses migration, invasion, and metastasis in non-small cell lung cancer in vitro and in vivo.

Non-small cell lung cancer (NSCLC) is a highly aggressive cancer with one of the most prevalent malignant tumors. Metastasis in NSCLC is the major cause of treatment failure and cancer-related deaths. Yes-associated protein (YAP) is a transcriptional coactivator regulated by the evolutionarily conserved Hippo signaling pathway that regulates organ size, growth, and regeneration. YAP is highly expressed in several malignant tumor types. Furthermore, YAP promotes tumor initiation and/or progression in various types of cancer. However, it is unclear whether YAP contributes to the metastasis in NSCLC and serves as a useful therapeutic target. Here, we investigated whether levels of YAP correlate with metastatic phenotype in NSCLC cells and serve as a useful therapeutic target. We found that high levels of YAP associate with high cell migration, invasion, and metastasis in NSCLC cell lines. Furthermore, YAP siRNA decreased the migration and invasion in NSCLC cells. Additionally, verteporfin, an agent used for the treatment of symptomatic polypoidal choroidal vasculopathy, decreased the expression of YAP and inhibited migration, invasion, and metastasis in NSCLC cells. Thus, the study suggests that targeting YAP may present a new avenue to develop therapeutics against metastasis in NSCLC and that verteporfin has potential molecular therapeutic strategy for the treatment of metastatic NSCLC.

Abstract 2838: Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state

Abstract Epigenetic dysregulations have a recognized role in cancer pathogenesis. Histone methyltransferase MLL3 (mixed-lineage leukemia protein 3) (gene symbol - KMT2C) is one of the most frequently altered genes in human breast cancer. In addition to the loss-of-function mutations, the expression levels of MLL3 are downregulated significantly in the tumor, compared to normal breast samples. Consistent with these cancer genome findings, functional studies in mouse models and human cancer cells showed loss of MLL3 promotes tumor initiation and growth. However, the role of MLL3 mutation in metastasis has not been determined. Here we showed that the MLL3 levels were further reduced in distant metastases compared to primary tumors, suggesting a potential role of MLL3 loss in metastasis. We demonstrate that MLL3 loss markedly promoted distant metastasis through specifically enhancing the step of colonization during the metastatic cascade. MLL3 loss facilitated the acquisition of an epithelial/mesenchymal (E/M) transition state by cancer cells disseminated to the lung. These cells have a hybrid phenotype of epithelial and mesenchymal features. We further showed that the E/M transitional cells induced by MLL3 loss have greatly increased tumor-initiating ability and multi-organ metastatic ability. We further demonstrated that MLL3 lose facilitated luminal breast tumor cells to acquire the E/M hybrid features and promoted endocrine therapy resistance, suggesting that loss of MLL3 also promotes the epithelial-mesenchymal plasticity and therapeutic resistance. To identify vulnerability of MLL3-mutant cancer cells, we performed small molecule screening and discovered that MLL3-mutant cancer cells are markedly more sensitive to BET inhibitors than MLL3-WT cells. The in vivo study confirmed that the BET inhibitor repressed the MLL3-mutant tumor growth much more efficiently than MLL3-WT tumors. Furthermore, the BET inhibitor is effective in suppressing the growth of established MLL3-mutant lung metastasis. Taken together, our findings indicate that MLL3 mutations promotes metastasis and therapeutic resistance by enhancing epithelial-mesenchymal plasticity. However, these aggressive cancer cells are uniquely vulnerable to BET inhibition, suggesting a potential therapeutic strategy for MLL3-mutant cancers. Citation Format: Jihong Cui, Kai Ge, Wenjun Guo. Epigenetic regulator MLL3 mutations promote tumor metastasis by enhancing the epithelial-mesenchymal transition state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2838.

From multi-omics integration towards novel genomic interaction networks to identify key cancer cell line characteristics

Cancer is a complex disease where cancer cells express epigenetic and transcriptomic mechanisms to promote tumor initiation, progression, and survival. To extract relevant features from the 2019 Cancer Cell Line Encyclopedia (CCLE), a multi-layer nonnegative matrix factorization approach is used. We used relevant feature genes and DNA promoter regions to construct genomic interaction network to study gene–gene and gene—DNA promoter methylation relationships. Here, we identified a set of gene transcripts and methylated DNA promoter regions for different clusters, including one homogeneous lymphoid neoplasms cluster. In this cluster, we found different methylated transcription factors that affect transcriptional activation of EGFR and downstream interactions. Furthermore, the hippo-signaling pathway might not function properly because of DNA hypermethylation and low gene expression of both LATS2 and YAP1. Finally, we could identify a potential dysregulation of the CD28-CD86-CTLA4 axis. Characterizing the interaction of the epigenome and the transcriptome is vital for our understanding of cancer cell line behavior, not only for deepening insights into cancer-related processes but also for future disease treatment and drug development. Here we have identified potential candidates that characterize cancer cell lines, which give insight into the development and progression of cancers.

Hypothyroidism induces oxidative stress and DNA damage in breast.

Breast cancer and thyroid dysfunctions have been associated for decades. Although many studies suggest a biological correlation, the mechanisms linking these two pathologies have not been elucidated. Reactive oxygen species (ROS) can oxidize lipids, proteins, and DNA molecules and may promote tumor initiation. Hence, we aimed at evaluating the mammary redox balance and genomic instability in a model of experimental hypothyroidism. Female Wistar rats were treated with 0.03% methimazole for 7 or 21 days to evaluate ROS generation, antioxidant enzyme activities, and oxidative stress biomarkers, as well as genomic instability. After 7 days, lower catalase, GPX, and DUOX activities were detected in the breast of hypothyroid group compared to the control while the levels of 4-hydroxynonenal (HNE) were higher. In addition, hypothyroid group showed an increase in γH2Ax/H2Ax ratio. Twenty-one days hypothyroid group had increased catalase and SOD activities, without significant differences between groups in the levels of oxidative stress biomarkers and DNA damage. TSH-treated MCF10A cells showed a higher extracellular, intracellular, and mitochondrial ROS production. Additionally, greater DNA damage was observed in these cells, demonstrated by a higher comet tail DNA percentage and increased 53BP1 foci. Finally, we found that TSH treatment was not able to alter cell viability. The Genome Cancer Atlas (TGCA) data showed that high TSHR expression is associated with more invasive breast cancer types. In conclusion, we demonstrate that oxidative stress and DNA damage in breast are early events of experimental hypothyroidism. Moreover, high TSH levels induce oxidative stress and genomic instability in mammary cells.