Tumor Suppressor Properties Research Articles

Oncogenic RUNX3: A Link between p53 Deficiency and MYC Dysregulation.

The RUNX transcription factors serve as master regulators of development and are frequently dysregulated in human cancers. Among the three family members, RUNX3 is the least studied, and has long been considered to be a tumor-suppressor gene in human cancers. This idea is mainly based on the observation that RUNX3 is inactivated by genetic/epigenetic alterations or protein mislocalization during the initiation of tumorigenesis. Recently, this paradigm has been challenged, as several lines of evidence have shown that RUNX3 is upregulated over the course of tumor development. Resolving this paradox and understanding how a single gene can exhibit both oncogenic and tumor-suppressive properties is essential for successful drug targeting of RUNX. We propose a simple explanation for the duality of RUNX3: p53 status. In this model, p53 deficiency causes RUNX3 to become an oncogene, resulting in aberrant upregulation of MYC.

Gut Microbiota and Cancer of the Host: Colliding Interests.

Cancer develops in multicellular organisms from cells that ignore the rules of cooperation and escape the mechanisms of anti-cancer surveillance. Tumorigenesis is jointly encountered by the host and microbiota, a vast collection of microorganisms that live on the external and internal epithelial surfaces of the body. The largest community of human microbiota resides in the gastrointestinal tract where commensal, symbiotic and pathogenic microorganisms interact with the intestinal barrier and gut mucosal lymphoid tissue, creating a tumor microenvironment in which cancer cells thrive or perish. Aberrant composition and function of the gut microbiota (dysbiosis) has been associated with tumorigenesis by inducing inflammation, promoting cell growth and proliferation, weakening immunosurveillance, and altering food and drug metabolism or other biochemical functions of the host. However, recent research has also identified several mechanisms through which gut microbiota support the host in the fight against cancer. These mechanisms include the use of antigenic mimicry, biotransformation of chemotherapeutic agents, and other mechanisms to boost anti-cancer immune responses and improve the efficacy of cancer immunotherapy. Further research in this rapidly advancing field is expected to identify additional microbial metabolites with tumor suppressing properties, map the complex interactions of host-microbe 'transkingdom network' with cancer cells, and elucidate cellular and molecular pathways underlying the impact of specific intestinal microbial configurations on immune checkpoint inhibitor therapy.

FAM46C suppresses gastric cancer by inhibition of Wnt/beta-catenin.

Gastric cancer (GC) is the most common cancer with a poor prognosis and the third leading cause of cancer death in the world, for which no effective therapeutic target exists. We tested the hypothesis that FAM46C might be involved in regulation of proliferation and apoptosis in GC. FAM46C was down-regulated and its expression negatively correlated with the expression of b-catenin that drives proliferation and apoptosis. Overexpression of FAM46C inhibited cell proliferation, induced G1 phase arrest and promoted apoptosis. Activation of Wnt/b-catenin signaling pathway in GC cell lines quenched the effect of FAM46C overexpression. On the other hand, FAM46C silencing attenuated DKK1-mediated inhibition of G1 phase, cessation of proliferation and induction apoptosis. Together, these data show that FAM46C shows tumor suppressor properties and such effects are mediated, at least in part, by Wnt/b-catenin in GC.

Connexin43 peptide, TAT-Cx43266-283, selectively targets glioma cells, impairs malignant growth, and enhances survival in mouse models in vivo.

BackgroundMalignant gliomas are the most frequent primary brain tumors and remain among the most incurable cancers. Although the role of the gap junction protein, connexin43 (Cx43), has been deeply investigated in malignant gliomas, no compounds have been reported with the ability to recapitulate the tumor suppressor properties of this protein in in vivo glioma models.MethodsTAT-Cx43266–283 a cell-penetrating peptide which mimics the effect of Cx43 on c-Src inhibition, was studied in orthotopic immunocompetent and immunosuppressed models of glioma. The effects of this peptide in brain cells were also analyzed.ResultsWhile glioma stem cell malignant features were strongly affected by TAT-Cx43266–283, these properties were not significantly modified in neurons and astrocytes. Intraperitoneally administered TAT-Cx43266–283 decreased the invasion of intracranial tumors generated by GL261 mouse glioma cells in immunocompetent mice. When human glioma stem cells were intracranially injected with TAT-Cx43266–283 into immunodeficient mice, there was reduced expression of the stemness markers nestin and Sox2 in human glioma cells at 7 days post-implantation. Consistent with the role of Sox2 as a transcription factor required for tumorigenicity, TAT-Cx43266–283 reduced the number and stemness of human glioma cells at 30 days post-implantation. Furthermore, TAT-Cx43266–283 enhanced the survival of immunocompetent mice bearing gliomas derived from murine glioma stem cells.ConclusionTAT-Cx43266–283 reduces the growth, invasion, and progression of malignant gliomas and enhances the survival of glioma-bearing mice without exerting toxicity in endogenous brain cells, which suggests that this peptide could be considered as a new clinical therapy for high-grade gliomas.

Bioinformatic Analysis and in Vitro Validation of Let-7b and Let-7c in Breast Cancer

Members of the let-7 family of miRNAs are well-known with their tumor suppressor properties as they are expressed at low levels in several types of human malignancies. Among them, let-7b and let-7c have gained special attention due their broad significance. Although the role of let-7b and let-7c have been widely reported in various types of cancers, their functional importance and role in oncogenic signaling of breast cancer is poorly investigated. Therefore, in the present study, prognostic and diagnostic significance of let-7b and let-7c in breast cancer and the effects these miRNAs on genes involved in cancer progression were determined by using several bioinformatics analysis and validated in vitro mimic assays, respectively. Using data of TCGA, OncomiR and dbDEMC 2.0, overall expression analysis of let-7b and let-7c was performed. The effect of let-7b and let-7c on genes involved in cancer progression was investigated by mimic transfection assays. We found that both let-7b and let-7c were significantly altered in breast cancer and associated with the clinicopathological findings of patients. Additionally, both let-7b and let-7c significantly altered oncogenic signaling in breast cancer cells. Consequently, both miRNAs might have fundamental roles in breast cancer progression and can be considered as potential targets for breast cancer therapy and diagnosis.

Tumor suppressor properties of the small C-terminal domain phosphatases in non-small cell lung cancer.

Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions. However their role in NSCLC remains insufficiently understood. For the first time we revealed the suppressor function of CTDSPs leading to a significant growth slowdown and senescence of A549 lung adenocarcinoma (ADC) cells in vitro. Their tumor-suppressive activity can be realized through increasing the proportion of the active form of Rb protein dephosphorylated at Ser807/811, Ser780, and Ser795 (P<0.05) thereby negatively regulating cancer cell proliferation. Moreover, we observed that a frequent (84%, 39/46) and highly concordant (Spearman’s rank correlation coefficient (rs) = 0.53–0.62, P≤0.01) down-regulation of CTDSPs and RB1 is characteristic of primary NSCLC samples (n=46). A clear difference in their mRNA levels was found between lung ADCs with and without lymph node metastases, but not in squamous cell carcinomas (SCCs) (P≤0.05). Based on The Cancer Genome Atlas (TCGA) data and the results obtained using the CrossHub tool, we suggest that the well-known oncogenic cluster miR-96/182/183 could be a common expression regulator of CTDSPs. Indeed, according to our qPCR, the expression of CTDSPs negatively correlates with these miRs, but positively correlates with their intronic miR-26a/b. Our results reflect functional association of CTDSP1, CTDSP2, and CTDSPL, expand knowledge about their suppressor properties through Rb dephosphorylation and provide new insights into the regulation of NSCLC growth.

The Interplay Between Innate Lymphoid Cells and the Tumor Microenvironment.

The multifaceted roles of Innate Lymphoid Cells (ILC) have been widely interrogated in tumor immunity. Whereas, Natural Killer (NK) cells possess undisputable tumor-suppressive properties across multiple types of cancer, the other ILC family members can either promote or inhibit tumor growth depending on the environmental conditions. The differential effects of ILCs on tumor outcome have been attributed to the high degree of heterogeneity and plasticity within the ILC family members. However, it is now becoming clear that ILCs responses are shaped by their dynamic crosstalk with the different components of the tumor microenvironment (TME). In this review, we will give insights into the molecular and cellular players of the ILCs-TME interactions and we will discuss how we can use this knowledge to successfully harness the activity of ILCs for anticancer therapies.

6mer Seed Toxicity in Viral microRNAs.

SummaryMicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.

MYCN and PRC1 cooperatively repress docosahexaenoic acid synthesis in neuroblastoma via ELOVL2

BackgroundThe MYCN amplification is a defining hallmark of high-risk neuroblastoma. Due to irregular oncogenes orchestration, tumor cells exhibit distinct fatty acid metabolic features from non-tumor cells. However, the function of MYCN in neuroblastoma fatty acid metabolism reprogramming remains unknown.MethodsGas Chromatography-Mass Spectrometer (GC-MS) was used to find the potential target fatty acid metabolites of MYCN. Real-time PCR (RT-PCR) and clinical bioinformatics analysis was used to find the related target genes. The function of the identified target gene ELOVL2 on cell growth was detected through CCK-8 assay, Soft agar colony formation assay, flow Cytometry assay and mouse xenograft. Chromatin immunoprecipitation (ChIP) and Immunoprecipitation-Mass Spectrometer (IP-MS) further identified the target gene and the co-repressor of MYCN.ResultsThe fatty acid profile of MYCN-depleted neuroblastoma cells identified docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid with anti-tumor activity, significantly increased after MYCN depletion. Compared with MYCN single-copy neuroblastoma cells, DHA level was significantly lower in MYCN-amplified neuroblastoma cells. RT-PCR and clinical bioinformatics analysis discovered that MYCN interfered DHA accumulation via ELOVL fatty acid elongase 2 (ELOVL2) which is a rate-limiting enzyme of cellular DHA synthesis. Enforced ELOVL2 expression in MYCN-amplified neuroblastoma cells led to decreased cell growth and counteracted the growth-promoting effect of MYCN overexpression both in vitro and vivo. ELOVL2 Knockdown showed the opposite effect in MYCN single-copy neuroblastoma cells. In primary neuroblastoma, high ELOVL2 transcription correlated with favorable clinical tumor biology and patient survival. The mechanism of MYCN-mediated ELOVL2 inhibition contributed to epigenetic regulation. MYCN recruited PRC1 (Polycomb repressive complex 1), catalysed H2AK119ub (histone 2A lysine 119 monoubiquitination) and inhibited subsequent ELOVL2 transcription.ConclusionsThe tumor suppressive properties of DHA and ELOVL2 are repressed by the MYCN and PRC1 jointly, which suggests a new epigenetic mechanism of MYCN-mediated fatty acid regulation and indicates PRC1 inhibition as a potential novel strategy to activate ELOVL2 suppressive functions.

Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent

BackgroundA healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear.MethodsA mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1−/−) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system.ResultsThe gastric tumor incidence was higher in the SULT2B1−/− mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction.ConclusionsThe abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.

Abstract AP23: EARLY LOSS OF MONOUBIQUITYLATION OF HISTONE H2B ALTERS KEY IMMUNE SIGNALING PATHWAYS PROMOTING THE PROGRESSION OF HIGH-GRADE SEROUS OVARIAN CANCER

Abstract High-grade serous ovarian carcinoma (HGSOC) is the most common form of ovarian cancer, accounting for over 70% of all cases. HGSOC is frequently complicated by the inevitable development of therapeutic resistance, and thus remains an incurable disease. Recent insights supporting the fallopian tube epithelium (FTE) as the point of origin for HGSOC and the serous tubal intraepithelial carcinoma (STIC) as the precursor lesion for a majority of HGSOCs provide the necessary context to study the mechanisms that drive the development and progression of HGSOC. Understanding these key molecular processes is essential to inform the development of next generation therapies. In this study we investigated the role of the ubiquitin ligase RNF20 and histone H2B monoubiquitylation (H2Bub1) in serous tumorigenesis. H2Bub1, catalyzed largely by RNF20, is an epigenetic mark with tumor suppressor properties. The loss of RNF20/H2Bub1 has been linked with cancer progression. We used immunohistochemistry to characterize the expression of H2Bub1 in HGSOC and FTE precursors. We found that H2Bub1 is lost or downregulated in a large proportion of STICs and HGSOC tumors, implicating RNF20/H2Bub1 loss as an early event in serous tumorigenesis. Consistent with our findings, analysis of TCGA data shows that the majority of HGSOCs exhibit heterozygous loss of RNF20. Functionally, we demonstrate that shRNA-mediated knockdown of RNF20, with concomitant loss of H2Bub1, is sufficient to increase cell migration and clonogenic growth, both in 2D and 3D, of immortalized FTE cells. To understand the mechanisms underlying these effects, we performed ATAC-seq and RNA-seq in RNF20 knockdown cells. Interestingly, major changes were observed in a number of immune signaling pathways, providing early mechanistic insights for the observed oncogenic phenotypes. Using ELISA we confirmed the upregulation of IL6 and other cytokines in RNF20 knockdown cells. Additionally, we confirmed that the oncogenic phenotype, enhanced migration, is in part driven by the increased levels of IL6 in RNF20 and H2Bub1 knockdown cells which can be neutralized by using an anti-IL6 antibody. In summary, our study identifies that loss of H2Bub1 is an early epigenetic event in HGSOC that rewires certain immune signaling pathways and may represent unique therapeutic opportunities. Citation Format: Jagmohan Hooda, Marián Novak, Matthew P. Salomon, Emily MacDuffie, Melissa Song, Jenny Lester, Vinita Parkash, Beth Y. Karlan, Moshe Oren, Dave S. Hoon, and Ronny Drapkin. EARLY LOSS OF MONOUBIQUITYLATION OF HISTONE H2B ALTERS KEY IMMUNE SIGNALING PATHWAYS PROMOTING THE PROGRESSION OF HIGH-GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP23.

Interleukin-37 Inhibits Colon Carcinogensis During Chronic Colitis.

Inflammatory bowel disease increases the risk of developing colon cancer. Interleukin (IL-) 37 is a fundamental inhibitor of innate immunity by reducing systemic and local inflammation. IL-37 protein is expressed in healthy and diseased bowel and liver tissue. Here, we tested whether transgenic expression of human IL-37 protects IL-10 deficient (IL-10KO) mice from chronic colitis. IL-37tg mice were crossbred with IL-10KO mice. Homozygous IL-10KO/IL-37tg and IL10KO drank 2% dextran sulfate sodium (DSS) in water for 5 days to induce mild colitis. Colon carcinogenesis was triggered by intragastric administration of celecoxib. Endpoints were clinical parameters of colitis, cytokine responses in LPS-stimulated whole blood and explanted colon specimen and qPCR analysis of colon biopsies. Colon inflammation and number of adenoma—carcinoma were analyzed by histology. During the DSS-induction phase IL-10KO and IL-10KO/IL-37tg mice had a similar weight loss due to mild acute colitis. From day 115 there was a significantly improved weight gain in IL-10KO/IL37-tg mice, though colon length was similar. After ex vivo LPS stimulation whole blood of IL-10KO/IL-37tg compared to IL-10KO mice released less IL-6, IL-17, IFNγ, and TNFα and ex vivo colon cultures showed reduced IL-6 production both indicative of reduced inflammatory conditions under the influence of IL-37. Six out of 10 IL-10KO mice developed colon adenoma and carcinoma. Only one adenoma but no carcinoma was detected in colons of IL-10KO/IL-37tg mice. In conclusion, IL-37 transgene expression protects IL-10KO mice from colon carcinogenesis. It remains unclear whether IL-37 has direct tumor suppressing properties.

BCL11B regulates MICA/B-mediated immune response by acting as a competitive endogenous RNA.

Cancer immune surveillance is an important host protection process that inhibits carcinogenesis and maintains cellular homeostasis. The major histocompatibility complex class I-related molecules A and B (MICA and MICB) are NKG2D ligands that play important roles in tumor immune surveillance. In the present study, by a combined bioinformatics prediction and experimental approach, we identify BCL11B 3'-UTR as a putative MICA and MICB ceRNA. We demonstrate in several human cell lines of different origins that the knockdown of BCL11B downregulates surface expression of MICA and MICB. Furthermore, we demonstrate miRNA dependency of BCL11B-mediated MICA and MICB regulation in Dicer knockdown HCT116 cells. In addition, MICA/B-targeting miRNAs (miR-17, miR-93, miR-20a, miR-20b, miR-106a, and miR-106b) repressed the expression of BCL11B by targeting its 3'-UTR. Moreover, we showed that the BCL11B knockdown-mediated downregulation of MICA/B resulted in reduced NK cell elimination in vitro and in vivo through reduced recognition of NKG2D. Of particular significance, BCL11B displays tumor-suppressive properties. The expression of BCL11B is downregulated in colon cancer tissues and associated with a reduced median survival of colon cancer patients. Taken together, our study revealed a new mechanism of BCL11B that prevents immune evasion of cancerous cells by upregulation of the NKG2D ligands MICA and MICB in a ceRNA manner.

Tumor-suppressive circular RNAs: Mechanisms underlying their suppression of tumor occurrence and use as therapeutic targets.

Circular RNAs (circRNAs) have a covalently closed circular conformation and are structurally stable. Those circRNAs with tumor‐suppressive properties play an important role in tumorigenesis and metastasis and thus may be used as therapeutic targets of cancers. Herein, we review the current understanding of the classification of circRNAs and summarize the functions and mechanisms of circRNAs that have tumor‐suppressive roles in various cancers, including liver cancer (circARSP91, circADAMTS13, circADAMTS14, circMTO1, hsa_circ_0079299, and circC3P1), bladder cancer (circFNDC3B, circITCH, circHIPK3, circRNA‐3, cdrlas, and circLPAR1), gastric cancer (circLARP4, circYAP1, hsa_cric_0000096, hsa_circ_0000993, and circPSMC3), breast cancer (circ_000911, hsa_circ_0072309, and circASS1), lung cancer (hsa_circ_0000977, circPTK2, circ_0001649, hsa_circ_100395, and circ_0006916), glioma (circ_0001946, circSHPRH, and circFBXW7), and colorectal cancer (circITGA7 and hsa_circ_0014717). Thanks to their structural stability, these tumor‐suppressive circRNAs may be used as potential and potent therapeutic targets. Moreover, we propose a new method for the classification of circRNAs. Based on whether they can be translated, circRNAs can be divided into noncoding circRNAs and coding circRNAs.

EGFR-mediated autophagy in tumourigenesis and therapeutic resistance.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) that links extracellular signals to the control of cell survival, growth, proliferation and differentiation. Due to its frequent overexpression and hyperactivation, EGFR has been a therapeutic target for human malignancies. Unfortunately, the specialized inhibitors of EGFR or EGFR-mediated pathways have not yet achieved the desired clinical effects. Therefore, it is necessary to elucidate the EGFR-mediated molecular basis of tumourigenesis, development and therapeutic resistance and to identify potential therapeutic targets. Interestingly, emerging research has indicated that autophagy is closely related to tumourigenesis, tumour progression and chemoresistance. Both autophagy upregulation and downregulation have been observed in cancers, suggesting its dual oncogenic and tumour suppressor properties during malignant transformation. Importantly, EGFR has been demonstrated to be a critical determinant of whether autophagy has a cytoprotective or cytotoxic effect. Therefore, here, we mainly focus on the function of EGFR in autophagy, especially the potential mechanism. The EGFR-mediated pathways or proteins involved in autophagy regulation include (1) the EGFR-mTOR pathway; (2) the EGFR-RAS pathway; (3) EGFR-Beclin1; (4) the EGFR-STAT3 pathway and (5) EGFR-LAPTM4B (oncoprotein lysosomal-associated transmembrane protein 4B). In addition, we also describe the role of EGFR-mediated autophagy in chemoresistance and tumour therapy. We attempt to summarized the mechanism by which EGFR-mediated signalling pathways participate in regulating autophagy and to investigate how to use the existing knowledge to identify potential cancer therapeutic targets.

Roles and Regulation of Long Noncoding RNAs in Hepatocellular Carcinoma.

Next-generation sequencing has uncovered thousands of long noncoding RNAs (lncRNA). Many are reported to be aberrantly expressed in various cancers, including hepatocellular carcinoma (HCC), and play key roles in tumorigenesis. This review provides an in-depth discussion of the oncogenic mechanisms reported to be associated with deregulated HCC-associated lncRNAs. Transcriptional expression of lncRNAs in HCC is modulated through transcription factors, or epigenetically by aberrant histone acetylation or DNA methylation, and posttranscriptionally by lncRNA transcript stability modulated by miRNAs and RNA-binding proteins. Seventy-four deregulated lncRNAs have been identified in HCC, of which, 52 are upregulated. This review maps the oncogenic roles of these deregulated lncRNAs by integrating diverse datasets including clinicopathologic features, affected cancer phenotypes, associated miRNA and/or protein-interacting partners as well as modulated gene/protein expression. Notably, 63 deregulated lncRNAs are significantly associated with clinicopathologic features of HCC. Twenty-three deregulated lncRNAs associated with both tumor and metastatic clinical features were also tumorigenic and prometastatic in experimental models of HCC, and eight of these mapped to known cancer pathways. Fifty-two upregulated lncRNAs exhibit oncogenic properties and are associated with prominent hallmarks of cancer, whereas 22 downregulated lncRNAs have tumor-suppressive properties. Aberrantly expressed lncRNAs in HCC exert pleiotropic effects on miRNAs, mRNAs, and proteins. They affect multiple cancer phenotypes by altering miRNA and mRNA expression and stability, as well as through effects on protein expression, degradation, structure, or interactions with transcriptional regulators. Hence, these insights reveal novel lncRNAs as potential biomarkers and may enable the design of precision therapy for HCC.

MS05.03 Notch Signalling

The Notch signaling pathway is a highly conserved pathway that has a vital role in embryonic development and post-embryonic functions such as hematopoiesis, neural cell development and angiogenesis (1). The signaling pathway is activated by the interaction of 4 Notch receptors and their interactions with 5 different ligands. Signaling is characterized by juxtacrine interaction between ligands and receptors on neighboring cells or within the same cell. Alterations in this pathway have been detected in several tumors both as tumor promoter and tumor suppressor. Notch signaling does appear to have relevance in non-small cell lung cancers (NSCLC) (2). In approximately 30% of NSCLCs loss of Numb, a negative regulator of Notch1, leads to increased Notch activity and about 10% of NSCLCs have gain of function mutations in Notch1. A meta-analysis demonstrated that higher expression of Notch1 was correlated with more advanced tumors (3). In addition, higher expressions of both Notch1 and Notch3 were associated with poor prognosis (4,5). In pre-clinical studies inhibition of Notch3 signaling has reduced growth of lung cancers. In addition, it has been demonstrated that there is cross talk between Notch3 and EGFR pathways and inhibition of both pathways can induce expression of anti-apoptotic protein BIM. Finally, Notch signaling may have a role in induction of the epithelial to mesenchymal phenotype (EMT) in cancers (6). EMT is known to be associated with resistance to both cytotoxic agents and targeted agents and inhibition of Notch signaling can not only reverse EMT but also can enhance the anti-tumor activity of cytotoxic agents such as cisplatin and targeted agents such as erlotinib. The EMT phenotype is frequently observed in a sub-population of cancer cells with self-renewal capacity or cancer stem cells. Notch signaling may be crucial to survival of cancer stem cells and persistence of this population may contribute to resistance to therapeutic agents. In squamous cell lung cancers Notch signaling may have tumor suppressive properties (7). Loss of function mutations in Notch family of genes, predominantly in Notch receptors, are frequently identified in several squamous cell cancers including squamous cell cancer of the lung. Similarly, loss of function mutations in Notch genes, particularly Notch1 have been identified in small cell lung cancer (SCLC). Expression of Notch receptor in a mouse model of SCLC reduced tumor burden, suggesting its tumor suppressive properties. The expression of DLL3, one of the Notch ligands is induced in SCLC by a key transcription factor ASCL1.DLL3 is shown to downregulate Notch signaling in SCLCs and enhance the carcinogenic phenotype. All the above data suggest that Notch signaling is highly contextual. In some tumors this pathway may have tumor suppressive properties but in others tumor promoting properties. Defining the role of this pathway in tumor types may guide development of therapeutic strategies targeting the Notch signaling pathway. Bigas A, Espinosa L. The multiple usages of Notch signaling in development, cell differentiation and cancer. Curr Opin Cell Biol 2018;55:1-7. Westhoff B, Colaluca IN, D’Ario G, et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci 2009;106:22293-8. Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis. Sci Rep 2015;5:10338. Donnem T, Andersen S, Al-shibili K, et al. Prognostic impact of Notch ligands and receptors in nonsmall cell lung cancer: coexpression of Notch-1 and vascular endothelial growth factor-A predicts poor survival. Cancer 2010:116:5674-85. Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells. J Cancer Res Clin Oncol 2016;142:981-93. Yuan X, Wu H, Han N, et al. Notch signaling and EMT in non-small cell lung cancer: biological significance and therapeutic application. J Hematol Oncol 2014;7:87. Nowell CS, Radtke F. Notch as a tumor suppressor. Nat Rev Cancer 2017;17:145-159. squamous, Tumor suppressor, NOTCH

Inhibitory effects of Semaphorin 3F as an alternative candidate to anti-VEGF monoclonal antibody on angiogenesis

Vascular endothelial growth factor (VEGF) inhibition forms the basis for anti-angiogenic therapies. With the methods based on the monoclonal antibody-mediated typical VEGF blockade, pathological angiogenesis in the tumor microenvironment is inhibited and the limitation of tumor growth is provided; however, the existing tumor tissue cannot be intervened. In this study, the anti-angiogenic effects of Semaphorin (SEMA) 3F, which has frequently been reported to have tumor suppressive properties, on a chick chorioallantoic membrane model as well as in vitro cell-cell interactions were investigated and comparatively assessed using anti-VEGF antibody. Vascular endothelial cells and chick embryos were stimulated with 10-16ng/mL VEGF165 prior to SEMA 3F administration in order to generate pathological vascularization conditions. Both in vitro and in ovo results revealed that SEMA 3F suppressed VEGF165-induced abnormal vascularization more effectively than anti-VEGF. Moreover, the required dose of SEMA 3F was significantly lower than that of anti-VEGF (103 times less under in ovo conditions). In light of these results, SEMA 3F is recommended as an important therapeutic agent for the prevention of pathological angiogenesis. SEMA 3F may offer an effective and efficient anti-angiogenic intervention that can be administered at a lower dose alternative to typical VEGF blocking agents.

Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.

A Short SOX9 Peptide Mimics SOX9 Tumor Suppressor Activity and Is Sufficient to Inhibit Colon Cancer Cell Growth.

Differently from cytotoxic chemotherapies, targeted therapies do not necessarily drive cancer cells toward death, but reduce cell proliferation, angiogenesis, and/or prevent metastasis without affecting healthy cells. Oncogenic proteins that are hyperactivated and/or overexpressed in cancer cells are prime targets for such therapies. On the other hand, the activity of tumor suppressor proteins is more difficult to harness. Here, we identified a short SOX9 sequence (S9pep) located at the hinge between the HMG DNA-binding domain and the SOX-E central conserved domain that mimics SOX9 tumor-suppressive properties. Doxycycline-induced S9pep expression in DLD-1 colorectal cancer cells inhibited the growth potential of these cells, including colorectal cancer stem cells, restored cell-cell contact inhibition, and inhibited the activity of the oncogenic Wnt/β-catenin signaling pathway. It also significantly decreased tumor growth in BALB/cAnNCrl mice grafted with mouse doxycycline-inducible CT26 colorectal cancer cells in which S9pep was induced by treating them with doxycycline. As the Wnt/β-catenin signaling pathway is constitutively activated in 80% of colorectal cancer and SOX9-inactivating mutations are present in up to 11% of colorectal cancer, S9pep could be a promising starting point for the development of a peptide-based therapeutic approach to restore a SOX9-like tumor suppressor function in colorectal cancer.