Early Tumorigenesis Research Articles

An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions

ObjectiveCellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial...

Deep sequencing reveals the genomic characteristics of lung adenocarcinoma presenting as ground-glass nodules (GGNs).

BackgroundThe concept of multi-step progression from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma (ADC) has been proposed, and ground-glass nodules (GGNs) may play a critical role during the early lung tumorigenesis. We present the first comprehensive description of the genomic architecture of GGNs to unravel the genetic basis of GGN.MethodsWe investigated 30 GGN-like lungs ADC by performing >1,000× whole-exome sequencing (WES) and characterized the genomic variations and evaluate the relationship between the clinicopathologic and molecular characteristics in this disease.ResultsDespite the low somatic mutation burden, GGNs exhibited high intratumor heterogeneity (ITH) characterized by the proportion of subclonal mutations. Different mutagenesis shaped the genomes of GGN during cancer evolution and were mostly featured by molecular clock-like signatures that occur in clonal mutations and defective DNA mismatch signatures that occur in subclonal mutations. Moreover, 10.7–67.1% clonal mutations occurred after whole-genome doubling (WGD), indicating that WGD could be a frequent truncal event in GGNs. Samples with WGD showed higher genomic instability but lower ITH. These GGNs were characterized by recurrent focal copy-number changes that are highly associated with tumorigenesis, with only two genes (EGFR and RBM10) that were recurrently mutated. Additionally, GGNs with different pathological subtypes or computed tomography (CT) features exhibited distinct genetic characteristics. Lepidic predominant or pure GGNs in CT images carried a lower mutation burden and had a relatively stable genome than nonlepidic or mixed GGNs. GGNs with RBM10 mutations tended to accompany a pathologically lepidic pattern, indicating RBM10 may drive the distinct subtype of lung cancer with better prognosis.ConclusionsThese findings facilitated interpreting the genomic characteristics of GGNs, provided insight into the early stages of lung cancer evolution, and possessed potential clinical significance.

The deacetylation-phosphorylation regulation of SIRT2-SMC1A axis as a mechanism of antimitotic catastrophe in early tumorigenesis.

Improper distribution of chromosomes during mitosis can contribute to malignant transformation. Higher eukaryotes have evolved a mitotic catastrophe mechanism for eliminating mitosis-incompetent cells; however, the signaling cascade and its epigenetic regulation are poorly understood. Our analyses of human cancerous tissue revealed that the NAD-dependent deacetylase SIRT2 is up-regulated in early-stage carcinomas of various organs. Mass spectrometry analysis revealed that SIRT2 interacts with and deacetylates the structural maintenance of chromosomes protein 1 (SMC1A), which then promotes SMC1A phosphorylation to properly drive mitosis. We have further demonstrated that inhibition of SIRT2 activity or continuously increasing SMC1A-K579 acetylation causes abnormal chromosome segregation, which, in turn, induces mitotic catastrophe in cancer cells and enhances their vulnerability to chemotherapeutic agents. These findings suggest that regulation of the SIRT2-SMC1A axis through deacetylation-phosphorylation permits escape from mitotic catastrophe, thus allowing early precursor lesions to overcome oncogenic stress.

Elucidating the genetic architecture underlying IGF1 levels and its impact on genomic instability and cancer risk

Background: Insulin-like growth factor-1 (IGF1) has been implicated in mitogenic and anti-apoptotic mechanisms that promote susceptibility to cancer development and growth. Previous epidemiological studies have described phenotypic associations between higher circulating levels of IGF1 in adults with higher risks for breast, prostate, ovarian, colorectal, melanoma and lung cancers. However, such evidence is prone to confounding and reverse causality. Furthermore, it is unclear whether IGF1 promotes only the survival and proliferation of cancerous cells, or also the malignant transformation of healthy cells. Methods: We perform a genome-wide association study in 428,525 white European ancestry individuals in the UK Biobank study (UKBB) and identify 831 independent genetic determinants of circulating IGF1 levels, double the number previously reported. Results: Collectively these signals explain ~7.5% of the variance in circulating IGF1 levels in EPIC-Norfolk, with individuals in the highest 10% of genetic risk exhibiting ~1 SD higher levels than those in the lowest 10%. Using a Mendelian randomization approach, we demonstrate that genetically higher circulating IGF1 levels are associated with greater likelihood of mosaic loss of chromosome Y in leukocytes in men in UKBB (OR per +1 SD = 1.038 (95% CI: 1.010-1.067), P=0.008) and 23andMe, Inc. (P=6.8×10-05), a biomarker of genomic instability involved in early tumorigenesis. Genetically higher IGF1 is also associated with higher risks for colorectal (OR = 1.126 (1.048-1.210), P=1.3×10-03) and breast cancer (OR= 1.075 (1.048-1.103), P=3.9×10-08), with similar effects on estrogen positive (ER+) (OR = 1.069 (1.037-1.102), P=2.3×10-05) and estrogen negative (ER-) (OR = 1.074 (1.025-1.125), P=3.9×10-08) subtypes. Conclusions: These findings give an insight into the genetic regulation of circulating IGF1 levels and support a causal role for IGF1 in early tumorigenesis and risks for breast and colorectal cancers.

Immunohistochemical Assessment of TNFAIP3/A20 Expression Correlates With Early Tumorigenesis in Breast Cancer.

Limited data exist on the expression pattern of TNFAIP3/A20, as assayed by immunohistochemistry (IHC), in breast cancer tissues. This study aimed to assess A20 expression pattern in breast cancer. The expression of A20 was analysed using IHC in 50 breast cancer cases retrieved from the Sharjah Breast Cancer Center at the University Hospital Sharjah, United Arab Emirates. Omics survival data were also used to analyse its association with survival in endocrine-treated subgroups. A20 expression in breast cancer tissues was 'tumor-specific', and as compared to normal tissue areas, its expression was associated with both intensity and extent in early grade 1 (p<0.0001) in all molecular subtypes. In addition, using omics survival data from a cohort of 3,520 breast cancer patients, we showed that A20 overexpression associated with lower overall survival rate in the endocrine treated subgroups [hazard ratio (HR)=2.14, 95%CI=1.61-2.82, p<0.0001]. A20 can serve as a biomarker for early diagnosis of breast cancers.

Management of early lung tumorigenesis by corilagin via modulation of proliferating cell nuclear antigen and apoptotic pathway

Background: Corilagin is vastly found in plants and incorporated in countless traditional medicinal systems to treat various ailments including cancer disease. However, till now, no scientific study has been done to validate its efficiency with appropriate experimental evidence. Resulting, this study examined the ameliorative effects of corilagin in benzo(a)pyrene (B[a] P)-induced lung cancer in vivo. Methods: Experimentally, the B(a)P (50 mg/kg body weight [b.wt.]) was administered orally to initiate lung cancer in mice, and the supplementation of prophylactic and therapeutic treatment by corilagin (30 mg/kg b.wt.) was observed for changes in b.wt. and lung weight, tumor incidence, oxidative stress marker (lipid peroxidation), tumor markers (carcinoembryonic antigen, neuron-specific enolase, aryl hydrocarbon hydroxylase, lactate dehydrogenase, γ-glutamyl transpeptidase, 5′-nucleotidase, alpha-keto dehydrogenase, citric acid cycle enzymes isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase), histological and immunohistochemical analysis (proliferating cell nuclear antigen), and apoptotic mRNA gene expression (Bax, Bcl-2, caspase-3, caspase-9, and cytochrome-c). Results: B(a)P-induced animals exhibited unusual changes in b.wt. and lung weight, tumor incidence, oxidative stress marker, enzymatic antioxidants, tumor markers, histological lung tissue damage, number of proliferating cell nuclear antigen, and apoptotic mRNA gene expression. On prophylactic and therapeutic treatment of corilagin, the animals showed reinstatement of all the above alterations to the near normal. Conclusion: These findings of all the analyzed data demonstrate that the B(a)P-induced animals of pre- and post-treated with corilagin may prevent initiation of lung cancer, hence establishing its chemopreventive effect against lung tumorigenesis.

Paneth cell-derived growth factors support tumorigenesis in the small intestine.

Paneth cells (PCs) are small intestinal epithelial cells that secrete antimicrobial peptides and growth factors, such as Wnt ligands. Intriguingly, the context in which PC-derived Wnt secretion is relevant in vivo remains unknown as intestinal epithelial ablation of Wnt does not affect homeostatic proliferation or restitution after irradiation injury. Considering the importance of growth factors in tumor development, we explored here the role of PCs in intestinal carcinogenesis using a genetic model of PC depletion through conditional expression of diphtheria toxin-α subunit. PC depletion in Apc Min mice impaired adenoma development in the small intestine and led to decreased Wnt3 expression in small bowel adenomas. To determine if PC-derived Wnt3 was required for adenoma development, we examined tumor formation after PC-specific ablation of Wnt3 We found that this was sufficient to decrease small intestinal adenoma formation; moreover, organoids derived from these tumors displayed slower growth capacity. Overall, we report that PC-derived Wnt3 is required to sustain early tumorigenesis in the small bowel and identify a clear role for PC-derived Wnt production in intestinal pathology.

Progastrin production transitions from Bmi1+/Prox1+ to Lgr5high cells during early intestinal tumorigenesis

Progastrin is an unprocessed soluble peptide precursor with a well-described tumor-promoting role in colorectal cancer. It is expressed at small levels in the healthy intestinal mucosa, and its expression is enhanced at early stages of intestinal tumor development, with high levels of this peptide in hyperplastic intestinal polyps being associated with poor neoplasm-free survival in patients. Yet, the precise type of progastrin-producing cells in the healthy intestinal mucosa and in early adenomas remains unclear. Here, we used a combination of immunostaining, RNAscope labelling and retrospective analysis of single cell RNAseq results to demonstrate that progastrin is produced within intestinal crypts by a subset of Bmi1+/Prox1+/LGR5low endocrine cells, previously shown to act as replacement stem cells in case of mucosal injury. In contrast, our findings indicate that intestinal stem cells, specified by expression of the Wnt signaling target LGR5, become the main source of progastrin production in early mouse and human intestinal adenomas. Collectively our results suggest that the previously identified feed-forward mechanisms between progastrin and Wnt signaling is a hallmark of early neoplastic transformation in mouse and human colonic adenomas.

MOF Regulates TNK2 Transcription Expression to Promote Cell Proliferation in Thyroid Cancer.

MOF is a well-known histone acetyltransferase to catalyze acetylation of histone H4 lysine 16 (K16), and it is relevant to diverse biological processes, such as gene transcription, cell cycle, early embryonic development and tumorigenesis. Here, we identify MOF as an oncogene in most thyroid cancer. It is found that expression level of MOF was significantly upregulated in most thyroid cancer tissue samples and cell lines. MOF-deficient in both BHP-10-3 and TT2609 cell lines inhibited cell proliferation by blocking the cell cycle in G1 phase and enhanced cell apoptosis. Mechanistically, MOF bound the TNK2 promoter to activate TNK2 transcription. Furthermore, the expression level of TNK2 was decreased with the histone acetyltransferase inhibitor. Besides, MOF promoted proliferation of thyroid cancer cells through increased phosphorylation of AKT, thus activating the PI3K/AKT pathway. Ultimately, our findings indicated that MOF played an oncogene role in development and progression of thyroid cancer and may be a potential novel target for the treatment of thyroid cancer.

Triangular Relationship between p53, Autophagy, and Chemotherapy Resistance.

Chemotherapy and radiation often induce a number of cellular responses, such as apoptosis, autophagy, and senescence. One of the major regulators of these processes is p53, an essential tumor suppressor that is often mutated or lost in many cancer types and implicated in early tumorigenesis. Gain of function (GOF) p53 mutations have been implicated in increased susceptibility to drug resistance, by compromising wildtype anti-tumor functions of p53 or modulating key p53 processes that confer chemotherapy resistance, such as autophagy. Autophagy, a cellular survival mechanism, is initially induced in response to chemotherapy and radiotherapy, and its cytoprotective nature became the spearhead of a number of clinical trials aimed to sensitize patients to chemotherapy. However, increased pre-clinical studies have exemplified the multifunctional role of autophagy. Additionally, compartmental localization of p53 can modulate induction or inhibition of autophagy and may play a role in autophagic function. The duality in p53 function and its effects on autophagic function are generally not considered in clinical trial design or clinical therapeutics; however, ample pre-clinical studies suggest they play a role in tumor responses to therapy and drug resistance. Further inquiry into the interconnection between autophagy and p53, and its effects on chemotherapeutic responses may provide beneficial insights on multidrug resistance and novel treatment regimens for chemosensitization.

Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 &amp; MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ &amp; AX) and MIB2-6 t(11;14) (BC &amp; BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

RUNX3 methylation drives hypoxia-induced cell proliferation and antiapoptosis in early tumorigenesis

Inactivation of tumor suppressor Runt-related transcription factor 3 (RUNX3) plays an important role during early tumorigenesis. However, posttranslational modifications (PTM)-based mechanism for the inactivation of RUNX3 under hypoxia is still not fully understood. Here, we demonstrate a mechanism that G9a, lysine-specific methyltransferase (KMT), modulates RUNX3 through PTM under hypoxia. Hypoxia significantly increased G9a protein level and G9a interacted with RUNX3 Runt domain, which led to increased methylation of RUNX3 at K129 and K171. This methylation inactivated transactivation activity of RUNX3 by reducing interactions with CBFβ and p300 cofactors, as well as reducing acetylation of RUNX3 by p300, which is involved in nucleocytoplasmic transport by importin-α1. G9a-mediated methylation of RUNX3 under hypoxia promotes cancer cell proliferation by increasing cell cycle or cell division, while suppresses immune response and apoptosis, thereby promoting tumor growth during early tumorigenesis. Our results demonstrate the molecular mechanism of RUNX3 inactivation by G9a-mediated methylation for cell proliferation and antiapoptosis under hypoxia, which can be a therapeutic or preventive target to control tumor growth during early tumorigenesis.

Tepary bean (Phaseolus acutifolius) lectin fraction provokes reversible adverse effects on rats' digestive tract.

The Tepary bean (Phaseolus acutifolius) lectin fraction (TBLF) exhibits differential cytotoxicity on colon cancer cells and inhibition of early tumorigenesis in the colon (50mg/kg, three times per week, for 6weeks). TBLF showed low toxicity with the ability to activate the immune system; however, some adverse effects are the loss in body weight gain, intestinal atrophy, and pancreatic hyperplasia. After a recovery period of 2weeks after treatment, reversion of pancreatic hyperplasia but no recovery of intestinal atrophy was observed. As TBLF has shown anticancer effects on the colon, it is important to characterize the adverse effects and how they can be reversed. Sprague Dawley rats were administered with TBLF (50mg/kg) for 6weeks, three times per week, and then allowed to recover for 6weeks post-treatment. After TBLF administration, small intestine atrophy, villus atrophy, and cryptic hyperplasia were confirmed, as well as increased intestinal mucus production, increased permeability and a decrease in the apparent ileal digestibility of crude proteins. The colon showed damage in the simple prismatic tissue and decreased crypt depth, and changes in microbiota and a decrease in the apparent fecal digestibility of crude protein were determined. Our results show that the adverse effects provoked by TBLF were partially reversed after 6weeks of recovery post-treatment, suggesting that increasing the recovery period it could be possible to reverse all adverse effects observed.

Abstract 4899: Investigating mutation co-operativity in early tumorigenesis of low-grade serous ovarian carcinoma with organoid model system and single-cell RNA sequencing

Abstract Background: Ovarian cancers are the most common gynecologic malignancies. Low grade serous ovarian carcinoma (LGSOC) is a rare tumor, accounting for ~2000 cases diagnosed every year in North America. Most of LGSOCs are characterized by high fatality rates over the long term, with only 20% of women surviving 10 years after diagnosis, due suboptimal response to current chemotherapies. Understanding the molecular events is crucial for developing better early detection strategies and more informed therapeutic options. LGSOC harbors a relatively stable genome, with common activating mutations in BRAF, KRAS and NRAS. Recently, NRAS mutations (Q61R) were found to co-exist with EIF1AX mutations (G8E) in LGOSC, and the two mutated proteins functionally cooperate. Increasing histological and gene expression evidence suggest that the cell of origin of LGSOC is in the Fallopian tube. Low incidence of this disease means it is poorly understood, and the resulting lack of available models further limits the study of underlying mechanisms. We therefore propose to use organoid cultures. These consist of 3D multicellular units that resemble in vitro a tissue or organ of body, both structurally and functionally. Objective: to elucidate molecular events underpinning LGSOC, specifically how NRAS(Q61R) and EIF1AX (G8E) mutations co-operate to drive early stages of tumorigenesis, with organoid system and single-cell RNA sequencing (scRNA-seq) technologies. Method: To reflect genetic background and cell of origin of LGSOC, NRAS Q61R and EIF1AX G8E mutant proteins were overexpressed via lentiviral transduction in organoid cultures of normal human Fallopian tubes. After allowing organoids to establish, 2 weeks after transduction gene expression alterations were resolved with scRNA-seq. Histology of organoids were assessed for histomorphological signs of transformation. Patient-derived tumor organoids (PDTOs) were also cultured to assess how well our LGSOC-modelling organoids (LMOs) recapitulate the histological features of patient tumours. Result: LMOs showed cytologic signs of transformation such as increased nuclear/cytoplasmic ratio, prominent nucleoli, and cellular pleomorphism. Papillary structures, a major histologic characteristic of LGSOC tumor were also observed in LMOs. PDTOs showed similar cytological features and organization as LMOs. From scRNA-seq, we identified genes up-regulated in double-mutant compared to single-mutant organoids such as CA125 and TACSTD2. CA125 is one of the earliest identified biomarkers for ovarian cancer and has remained to be the most useful serum marker despite limited sensitivity and specificity; whereas TACSTD2 overexpression has been found to correlate with a chemo-resistant, aggressive malignant phenotype. Conclusion and future directions: Organoid culture and scRNA-seq is a powerful duo in studying early tumorigenesis events. We established a novel model system of LGSOC by introducing common co-occurring mutations into normal Fallopian tube tissues. Our model recapitulates to a large extent of LGSOC histology. Genes upregulated in double mutants included well-characterized biomarker (CA125) and a potential biomarker or therapeutic target (TACSTD2). Our work will be crucial for developing early detection strategies and targeted treatment options. Citation Format: Joyce Yu Han Zhang, Dawn Cochrane, Kieran Campbell, Minh Bui, Germain Ho, Cindy Shen, Winnie Yang, Clara Salamanca, Genny Trigo, David G. Huntsman. Investigating mutation co-operativity in early tumorigenesis of low-grade serous ovarian carcinoma with organoid model system and single-cell RNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4899.

Digital RNA Sequencing of Human Epidermal Keratinocytes Carrying Human Papillomavirus Type 16 E7

High-risk human papillomavirus (HPV) infections are the predominant cause of cervical cancer and its early gene E7 plays an important role in cellular proliferation and cell-cycle progression. While tremendous progress has been made in exploring the molecular mechanisms in late tumorigenesis, many pathways showing how HPV deregulates host gene expression in early inapparent infections and early tumorigenesis still remain undefined. Digital RNA sequencing was performed and a total of 195 differentially expressed genes were identified between the HPV16 E7-transfected NHEKs and control cells (p < 0.05, fold-change > 2). GO enrichment showed that HPV16 E7 primarily affected processes involved in anti-viral and immune responses, while KEGG pathway analysis showed enrichment of gene clusters of associated with HPV infection and MAPK signaling. Of the differentially expressed genes, IFI6, SLC39A9 and ZNF185 showed a strong correlation with tumor progression and patient survival in the OncoLnc database while roles for AKAP12 and DUSP5 in carcinogenesis and poor prognosis have previously been established for other cancer types. Our study identified several novel HPV16 E7-regulated candidate genes with putative functions in tumorigenesis, thus providing new insights into HPV persistence in keratinocytes and early onset of tumorigenesis.

Abstract A70: Studying the signaling pathways of tumorigenic epithelial fallopian tube subpopulations during early tumorigenesis

Abstract Survival rates for high-grade serous ovarian carcinoma (HGSOC) patients have not substantially improved over the past decades, mainly because the disease is only detected at the metastatic stage. The findings of early premalignant lesions in the fallopian tube, but not in the ovary, suggested that HGSOCs may originate from the fallopian tube epithelium (FTE) and metastasize to the ovary. Studying the mechanisms of HGSOC genesis from the correct cell of origin is critical to the early detection of the disease. Our main goal is to determine how genetic and molecular alterations contribute to the early events of HGSOC genesis from FTE. Deletion of PTEN alone in FTE is sufficient to lead to tumor formation and peritoneal dissemination, suggesting that loss of PTEN may play a pivotal role in the early events of the tumorigenic cascade. Herein we show that loss of PTEN in the FTE upregulates markers of tumor-initiating cells (TIC) such as WNT4, LGR5, ALDH1, c-Kit, CD44, and Dll4. We also discovered that the TIC markers are confined in a specific population of cells with increased diameter that we call TICHigh as compared to a smaller population that we call TICLow. In fact, when these two populations are separated by flow cytometry-based sorting, the TICHigh retains all the TIC markers. In addition, using a limiting dilution assay, we found that the TICHigh subpopulation forms tumor at a faster rate in vivo and colonize more the ovary ex vivo. In addition, TICHigh forms bigger spheroids and are more resistant to chemotherapeutic agents, suggesting that more targeted therapy may be developed for these stem-like, tumorigenic subpopulations. We also show that loss of PAX2 is mediating the TICHigh phenotype and that activation of AKT is not sufficient to induce the TIC phenotype whereas p70S6K activation is required. These findings suggest that therapies to re-express PAX2 may prevent development of HGSOC. Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis. Citation Format: Angela Russo, Matthew Dean, Dan Lantvit, Joanna E. Burdette. Studying the signaling pathways of tumorigenic epithelial fallopian tube subpopulations during early tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A70.

SPI1-induced upregulation of lncRNA SNHG6 promotes non-small cell lung cancer via miR-485-3p/VPS45 axis

More and more researches have proved that long noncoding RNAs (lncRNAs) are vital regulators and biological participants in human cancers [1–5]. SnoRNA host gene 6 (SNHG6) was found to have an effect on the early stage and tumorigenesis in many cancers [6–10]. However, the expression of SNHG6 and its role of in non-small cell lung cancer (NSCLC) still need to be investigated. This work aims to investigate the expression and its biological role in NSCLC. In our study, the expression of SNHG6 was abnormally high in NSCLC tissues and cells. The negative impact of SNHG6 expression on the overall survival of patients with NSCLC was analyzed with Kaplan Meier method. Functionally, loss of SNHG6 expression led to the inhibition on the growth, migration and invasion of NSCLC cells. Mechanistically, miR-485-3p was necessary for the regulatory relation between SNHG6 and VPS45. More importantly, SPI1 could promote the expression of SNHG6 via transcriptionally activation. In conclusion, we proved that SPI1/SNHG6/miR-485-3p/VPS45 axis exerted oncogenic role in the cellular process of NSCLC cells.

Multi-cancer analysis of clonality and the timing of systemic spread in paired primary tumors and metastases.

Metastasis is the primary cause of cancer-related deaths, but the natural history, clonal evolution and impact of treatment are poorly understood. We analyzed whole-exome sequencing data from 457 paired primary tumor and metastatic samples from 136 breast, colorectal and lung cancer patients, including untreated (n=99) and treated (n=100) metastases. Treated metastases often harbored private ‘driver’ mutations whereas untreated metastases did not, suggesting that treatment promotes clonal evolution. Polyclonal seeding was common in untreated lymph node metastases (n=17/29, 59%) and distant metastases (n=20/70, 29%), but less frequent in treated distant metastases (n=9/94, 10%). The low number of metastasis-private clonal mutations is consistent with early metastatic seeding, which we estimated occurred 2–4 years prior to diagnosis across these cancers. Further, these data suggest that the natural course of metastasis is selectively relaxed relative to early tumorigenesis and that metastasis-private mutations are not drivers of cancer spread but instead associated with drug resistance.

Differential levels of Alpha-1-inhibitor III, Immunoglobulin heavy chain variable region, and Hypertrophied skeletal muscle protein GTF3 in rat mammary tumorigenesis

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy.