Inhibit Tumor Initiation Research Articles

Thyroid Hormone Receptors as Tumor Suppressors in Cancer.

Accumulated research has revealed the multifaceted roles of thyroid hormone receptors (TRs) as potent tumor suppressors across various cancer types. This review explores the intricate mechanisms underlying TR-mediated tumor suppression, drawing insights from preclinical mouse models and cancer biology. This review examines the tumor-suppressive functions of TRs, particularly TRβ, in various cancers using preclinical models, revealing their ability to inhibit tumor initiation, progression, and metastasis. Molecular mechanisms underlying TR-mediated tumor suppression are discussed, including interactions with oncogenic signaling pathways like PI3K-AKT, JAK-STAT, and transforming growth factor β. Additionally, this paper examines TRs' effect on cancer stem cell activity and differentiation, showcasing their modulation of key cellular processes associated with tumor progression and therapeutic resistance. Insights from preclinical studies underscore the therapeutic potential of targeting TRs to impede cancer stemness and promote cancer cell differentiation, paving the way for precision medicine in cancer treatment and emphasizing the potential of TR-targeted therapies as promising approaches for treating cancers and improving patient outcomes.

DFMO inhibition of neuroblastoma tumorigenesis.

Most high-risk neuroblastoma patients who relapse succumb to disease despite the existing therapy. We recently reported increased event-free and overall survival in neuroblastoma patients receiving difluoromethylornithine (DFMO) during maintenance therapy. The effect of DFMO on cellular processes associated with neuroblastoma tumorigenesis needs further elucidation. Previous studies have shown cytotoxicity with IC50 values >5-15 mM, these doses are physiologically unattainable in patients, prompting further mechanistic studies at therapeutic doses. We characterized the effect of DFMO on cell viability, cell cycle, apoptosis, neurosphere formation, and protein expression invitro using five established neuroblastoma cell lines (BE2C, CHLA-90, SHSY5Y, SMS-KCNR, and NGP) at clinically relevant doses of 0, 50, 100, 500, 1000, and 2500 μM. Limiting Dilution studies of tumor formation in murine models were performed. Statistical analysis was done using GraphPad and the level of significance set at p = 0.05. There was not a significant loss of cell viability or gain of apoptotic activity in the invitro assays (p > 0.05). DFMO treatment initiated G1 to S phase cell cycle arrest. There was a dose-dependent decrease in frequency and size of neurospheres and a dose-dependent increase in beta-galactosidase activity in all cell lines. Tumor formation was decreased in xenografts both with DFMO-pretreated cells and in mice treated with DFMO. DFMO treatment is cytostatic at physiologically relevant doses and inhibits tumor initiation and progression in mice. This study suggests that DFMO, inhibits neuroblastoma by targeting cellular processes integral to neuroblastoma tumorigenesis at clinically relevant doses.

Abstract 425: GPD1L participates in glycerol-3-phosphate biogenesis and promotes tumor initiation and invasion in hepatocellular carcinoma

Abstract Background: Metabolic reprogramming in hepatocellular carcinoma (HCC) has received rising interest. However, current studies often focus on fatty acid de novo synthesis or oxidation, but less on glycerolipids metabolism. Interestingly, in HCC, we observed frequent dysregulation in enzymes responsible for glycerol-3-phosphate shuttle, which potentially links glycolysis and glycerolipids metabolism. Hence, we aimed to identify the functional significance of glycerol-3-phosphate dehydrogenase 1 like (GPD1L), an enzyme for the shuttle, and its regulation of expression in HCC. Methods: We utilized transcriptomic analysis of clinical HCC samples to validate the frequent overexpression of GPD1L in tumor cells and examined its association with clinicopathological features. In vitro and in vivo assays were performed to explore the functional significance of GPD1L in HCC initiation and progression. Furthermore, with targeted metabolomics analysis, we investigated the role of GPD1L in glycerol-3-phosphate shuttle in HCC. We also performed promoter study to understand the transcriptional regulation on GPD1L expression. Results: Among GPD family members, GPD1L was consistently upregulated at mRNA level in clinical HCC samples in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) (N=50) and our in-house RNA-sequencing cohorts (N=41), and in a separate independent in-house cohort as examined by RT-qPCR (N=80). GPD1L overexpression was associated with increased tumor invasiveness (p=0.008) and poorer overall survival (p=0.0017) in HCC patients. Functionally, knockdown of GPD1L suppressed HCC migration (fold change ≤ 0.43) and invasion (fold change ≤ 0.40). GPD1L knockdown also inhibited tumor initiation abilities, as shown by reduced colony (fold change ≤ 0.37) and sphere formation (fold change ≤ 0.41) in vitro, and lower stem cell frequency (p=1.96e-9) in vivo. Furthermore, GPD1L knockdown enhanced sensitivity of HCC cells to chemotherapeutic drugs, sorafenib and doxorubicin. Metabolically, GPD1L facilitated synthesis of glycerolipids precursor, glycerol-3-phosphate (G3P) from glycolysis intermediate dihydroxyacetone phosphate (DHAP) in HCC. Lastly, with chromatin-immunoprecipitation and Luciferase reporter assays, we demonstrated ELF1 bound to GPD1L promoter directly to enhance the gene transcription, while knockdown of ELF1 suppressed GPD1L mRNA and protein expression. Conclusion: Our study demonstrated the overexpression of the glycolysis-related gene, GPD1L, in HCC. GPD1L promoted tumor initiation, invasion and chemoresistance, and in turn might lead to worse clinical outcome. Metabolically, it was responsible for DHAP-to-G3P conversion in HCC and might serve as a linkage between glycolysis and glycerolipid metabolism. Our results also identified the novel role of ELF1 as the transcriptional activator of GPD1L in HCC. Citation Format: Tiffany Ching-Yun Yu, Luqing Zhao, Lu Tian, Charles Shing Kam, Joyce Man-Fong Lee, Daniel Wai-Hung Ho, Lo-Kong Chan, Yu-Man Tsui, Karen Man-Fong Sze, Irene Oi-Lin Ng. GPD1L participates in glycerol-3-phosphate biogenesis and promotes tumor initiation and invasion in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 425.

The Humanization and Maturation of an Anti-PrPc Antibody.

The cellular prion protein (PrPc) is a cell surface glycoprotein that is highly expressed in a variety of cancer tissues in addition to the nervous system, and its elevated expression is correlated to poor prognosis in many cancer patients. Our team previously found that patients with colorectal cancer (CRC) with high-level PrPc expression had significantly poorer survival than those with no or low-level PrPc expression. Mouse antibodies for PrPc inhibited tumor initiation and liver metastasis of PrPc-positive human CRC cells in mouse model experiments. PrPc is a candidate target for CRC therapy. In this study, we newly cloned a mouse anti-PrPc antibody (Clone 6) and humanized it, then affinity-matured this antibody using a CHO cell display with a peptide antigen and full-length PrPc, respectively. We obtained two humanized antibody clones with affinities toward a full-length PrPc of about 10- and 100-fold of that of the original antibody. The two humanized antibodies bound to the PrPc displayed significantly better on the cell surface than Clone 6. Used for Western blotting and immunohistochemistry, the humanized antibody with the highest affinity is superior to the two most frequently used commercial antibodies (8H4 and 3F4). The two new antibodies have the potential to be developed as useful reagents for PrPc detection and even therapeutic antibodies targeting PrPc-positive cancers.

Transformed cells after senescence give rise to more severe tumor phenotypes than transformed non-senescent cells

Oncogenic stress-induced senescence initially inhibits tumor initiation by blocking proliferation and by attracting immune cells to clear potentially harmful cells. If these cells are not eliminated they may resume proliferation upon loss-of-tumor suppressors, and be at risk of transformation. During tumor formation, depending on the sequence of events of gain-of-oncogenes and/or loss-of-tumor suppressors, cancer cells may emerge from senescent cells. Here, we show that these transformed cells after senescence (TS) display more aggressive tumorigenic features, with a greater capacity to migrate and a higher resistance to anti-tumoral drugs than cells having undergone transformation without senescence. Bulk transcriptomic analysis and single cell RNA sequencing revealed a signature unique to TS cells. A score of this signature was then generated and a high score was correlated with decreased survival of patients with lung adenocarcinoma, head-neck squamous cell carcinoma, adrenocortical carcinoma, liver hepatocellular carcinoma, skin cutaneous melanoma and low-grade glioma. Together, these findings strongly support that cancer cells arising from senescent cells are more dangerous, and that a molecular signature of these cells may be of prognostic value for some human cancers. It also raises questions about modeling human tumors, using cells or mice, without regards to the sequence of events leading to transformation.

PDGFA-associated protein 1 is a novel target of c-Myc and contributes to colorectal cancer initiation and progression.

BackgroundThe mechanism underlying colorectal cancer (CRC) initiation and progression remains elusive, and overall survival is far from satisfactory. Previous studies have shown that PDGFA‐associated protein 1 (PDAP1) is upregulated in several cancers including CRC. Here, we aimed to identify the cause and consequence of PDAP1 dysregulation in CRC and evaluate its role as a potential therapeutic target.MethodsMulti‐omics data analysis was performed to identify potential key players in CRC initiation and progression. Immunohistochemistry (IHC) staining was applied to determine the expression pattern of PDAP1 in CRC tissues. Pdap1 conditional knockout mice were used to establish colitis and CRC mouse models. RNA sequencing, a phosphoprotein antibody array, western blotting, histological analysis, 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and interactome analysis were applied to identify the underlying mechanisms of PDAP1. A human patient‐derived xenograft (PDX) model was used to assess the potential of PDAP1 as a therapeutic target.ResultsPDAP1 was identified as a potential key player in CRC development using multi‐omics data analysis. PDAP1 was overexpressed in CRC cells and correlated with reduced overall survival. Further investigation showed that PDAP1 was critical for the regulation of cell proliferation, migration, invasion, and metastasis. Significantly, depletion of Pdap1 in intestinal epithelial cells impaired mucosal restitution in dextran sulfate sodium salt‐induced colitis and inhibited tumor initiation and growth in colitis‐associated cancers. Mechanistic studies showed that c‐Myc directly transactivated PDAP1, which contributed to the high PDAP1 expression in CRC cells. PDAP1 interacted with the juxtamembrane domain of epidermal growth factor receptor (EGFR) and facilitated EGFR‐mitogen‐activated protein kinase (MAPK) signaling activation, which resulted in FOS‐related antigen 1 (FRA‐1) expression, thereby facilitating CRC progression. Notably, silencing of PDAP1 could hinder the growth of patient‐derived xenografts that sustain high PDAP1 levels.ConclusionsPDAP1 facilitates mucosal restitution and carcinogenesis in colitis‐associated cancer. c‐Myc‐driven upregulation of PDAP1 promotes proliferation, migration, invasion, and metastasis of CRC cells via the EGFR‐MAPK‐FRA‐1 signaling axis. These findings indicated that PDAP1 inhibition is warranted for CRC patients with PDAP1 overexpression.

Aspirin and celecoxib revealed anticancer and immunomodulatory effects on colon cancer cells

Colorectal cancer is one of the most common malignancies in the world. Clinical evidence suggests that non-steroidal anti-inflammatory drugs (NSAIDs ) were found to reduce the risk of colonic adenoma and colorectal cancer occurrence or recurrence and are more beneficial in acute inflammatory disorders than in chronic inflammatory diseases, implying that NSAIDs target the early stages of the inflammatory response. Celecoxib inhibits tumor initiation and tumor cell proliferation. Aspirin inhibits the constitutive isoform of the platelet enzyme cyclooxygenase- 1 (COX-1) and the inducible isoform cyclooxygenase-2 (COX-2) which is expressed by cytokines, and some growth factors. In this study, different concentrations of both Celecoxib and Aspirin in various sets were applied to investigate their effects on colon cancer cells (SW480) proliferation and cytokines production. Our result reported a significant decrease in cells viability after the treatment with aspirin and celecoxib alone or in combination. Celecoxib significantly decreased IL-6 levels, while Aspirin treatment showed no significant change in IL-6, IL-12, and TNF- α levels. In conclusion, non-steroidal anti-inflammatory drugs (NSAIDs) have pronounced anti-proliferative effects on colorectal cancer cell lines.

Macrophage cytotoxic activity and its role in the tumor pathogenesis

Macrophages, natural killers and T cells play the central role in tumor cells destruction. The purpose of this review is to summarize the state-of-the-art perspectives of the interplay between tumor cells and tumor stroma leading both to the formation of a macrophage population incapable of effective antitumor activity and to the selection of tumor cells resistant to macrophage cytotoxicity. Macrophages are highly versatile cells that can both stimulate the inflammatory response (type 1 macrophages, M1) and suppress it (type 2 macrophages, M2). Tumor-associated macrophages (TAMs) are considered the main regulator of the antitumor immune response and usually have anti-inflammatory properties, that is, they belong to M2 type. Tumor cells are able to affect macrophages, "reprogramming" them to perform an immunosuppressive function. In addition, TAMs stimulate angiogenesis and remodelling of the extracellular matrix necessary for metastasis. Recently, more and more studies have been published describing a mixed TAMs phenotype with characteristics of both M2 and M1. M1 is characterized by production of pro-inflammatory cytokines, reactive oxygen species, bactericidal and cytotoxic activity. M1 can destroy tumor cells both directly and indirectly by attracting other cells. Despite the mechanisms of direct cytotoxic activity are quite variable, their effectiveness is largely dependent on the properties of a particular tumor. The cytotoxic activity of macrophages is a powerful factor that inhibits tumor initiation and progression. However, in some cases, it is not sufficient to control the tumor process. Activation of the cytotoxic activity of TAMs is one of the strategies to use macrophages for cancer treatment. Understanding the mechanisms of macrophage cytotoxic activity and specific patterns of its manifestation in a tumor environment is of critical importance for better effectiveness of existing cancer treatments and development of promising methods for tumor immunotherapy.

ASCL1 represses a SOX9+ neural crest stem-like state in small cell lung cancer.

ASCL1 is a neuroendocrine lineage-specific oncogenic driver of small cell lung cancer (SCLC), highly expressed in a significant fraction of tumors. However, ∼25% of human SCLC are ASCL1-low and associated with low neuroendocrine fate and high MYC expression. Using genetically engineered mouse models (GEMMs), we show that alterations in Rb1/Trp53/Myc in the mouse lung induce an ASCL1+ state of SCLC in multiple cells of origin. Genetic depletion of ASCL1 in MYC-driven SCLC dramatically inhibits tumor initiation and progression to the NEUROD1+ subtype of SCLC. Surprisingly, ASCL1 loss promotes a SOX9+ mesenchymal/neural crest stem-like state and the emergence of osteosarcoma and chondroid tumors, whose propensity is impacted by cell of origin. ASCL1 is critical for expression of key lineage-related transcription factors NKX2-1, FOXA2, and INSM1 and represses genes involved in the Hippo/Wnt/Notch developmental pathways in vivo. Importantly, ASCL1 represses a SOX9/RUNX1/RUNX2 program in vivo and SOX9 expression in human SCLC cells, suggesting a conserved function for ASCL1. Together, in a MYC-driven SCLC model, ASCL1 promotes neuroendocrine fate and represses the emergence of a SOX9+ nonendodermal stem-like fate that resembles neural crest.

Concerted cell and in vivo screen for pancreatic ductal adenocarcinoma (PDA) chemotherapeutics

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

Targeting Autophagy to Treat Cancer: Challenges and Opportunities.

Autophagy is a catabolic process that targets its cargo for lysosomal degradation. In addition to its function in maintaining tissue homeostasis, autophagy is recognized to play a context-dependent role in cancer. Autophagy may inhibit tumor initiation under specific contexts; however, a growing body of evidence supports a pro-tumorigenic role of this pathway in established disease. In this setting, autophagy drives treatment resistance, metabolic changes, and immunosuppression both in a tumor-intrinsic and extrinsic manner. This observation has prompted renewed interest in targeting autophagy for cancer therapy. Novel genetic models have proven especially insightful, revealing unique and overlapping roles of individual autophagy-related genes in tumor progression. Despite identification of pharmacologically actionable nodes in the pathway, fundamental challenges still exist for successful therapeutic inhibition of autophagy. Here we summarize the current understanding of autophagy as a driver of resistance against targeted and immuno-therapies and highlight knowledge gaps that, if addressed, may provide meaningful advances in the treatment of cancer.

Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell-enriched Population by Stimulating Their Differentiation.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell–like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell–like cells harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed the genes important for myometrium- and LM-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5′-Aza, stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of LM tumors.

Abstract 2978: Cyclic GMP-independent inhibition of colon cancer cell growth by phosphodiesterase inhibitors

Abstract There is growing interest in the potential use of phosphodiesterase (PDE) inhibitors for colon cancer chemoprevention, but the tumor suppressive mechanism is not understood. The present study has tested the idea that PDE inhibitors can suppress growth and promote apoptosis of colon cancer cell lines by increasing cGMP and activating cGMP-dependent protein kinase (PKG). Treatment of LS174T, Caco2, and HCT116 colon cancer cells with membrane permeable 8Br-cGMP had no effect on cell proliferation or viability. Endogenous PKG was undetectable in any of these cells using phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (VASP) as a readout. Ectopic expression of PKG2 conferred modest inhibition of proliferation by cGMP treatment (15-25% over 3 days), but did not affect cell viability. The lack of effect of cGMP on colon cancer proliferation is incongruous with many reported effects of PDE inhibitors. To understand the reason for the discrepant observations, colon cancer cells were treated with sildenafil, PF-0447943, and TAK-063 that are specific inhibitors of the cGMP-targeting PDE5, 9, 10a (respectively). At clinically unachievable concentrations exceeding 10μM, all of these drugs reduced proliferation to various degrees in the different cell lines. TAK-063 was the most effective, and the only one that reduced cell viability. Notably, growth-inhibition by the PDE inhibitors was independent of PKG expression, and surprisingly, none of them increased cGMP levels. The inability of the PDE inhibitors to increase cGMP was due to the lack of endogenous cGMP generation, because treatment with the guanylyl-cyclase C agonist linaclotide conferred a robust cGMP response to a physiological dose of sildenafil (1µM). The growth inhibitor effects of high, non-physiological levels of PDE inhibitors is likely due to off-target effects, but apart from a transient inhibition of the ERK pathway, no common mechanism was identified. Taken together, these results refute the idea that cGMP elevating agents prevent colon carcinogenesis by inhibiting cancer cell growth, and support the inhibition of tumor initiation as a more likely mechanism. Citation Format: Bianca N. Islam, Yali Hou, Namratha Mylarapu, Kaylin A. Browning, Kenneth J. Vega, Darren D. Browning. Cyclic GMP-independent inhibition of colon cancer cell growth by phosphodiesterase inhibitors [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 2978.

Abstract 4794: Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells

Abstract Background: We previously reported that ONC201, a novel anti-tumor drug currently being tested in early phase clinical trials, impairs mitochondrial structure and function in breast cancer cells (Greer et al., 2018). Recent studies demonstrated that ONC201 and the related TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein to mitochondrial protein homeostasis (Graves et al. 2019). Cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we tested the impact of ClpP agonists-mediated mitochondrial dysregulation on breast CSCs. Methods: Mitochondrial DNA (mtDNA)-depleted (rho0) MDA-MB231 (MB231) cells were generated by ethidium bromide treatment. MtDNA copy numbers were measured by qPCR. Mitochondrial respiration was monitored by Seahorse XF analyzer. Cell viability was analyzed by CellTiter-Glo 2.0 and RealTime-Glo assay. Protein expression was examined by Western blotting. ClpP knockout (KO) cells were generated by CRISPR/Cas9 technology. Mammosphere formation assay was performed to evaluate CSC function. MB231 cells stably expressing SORE6 (OCT4/SOX2 response element)-mCherry reporter gene were used to monitor CSC numbers. In vivo limiting dilution analysis was performed to examine the effect of ClpP agonists on MB231 mammary fat pad tumor initiation capability in athymic nude female mice. Results: We observed that mammospheres formed by MB231were enriched in mtDNA compared to cells grown in 2D culture while rho0 cells lacking functional mitochondria failed to form mammospheres. This suggested that mitochondria are required for CSC function. The effects of ONC201 and TR compounds in breast cancer cells were tested. First, we confirmed that TR compounds (TR-65, TR-57) had ~60-250 fold higher potency compared to ONC201 in cell viability assays. Both ONC201 and TR-57 impaired mitochondrial structure, function, inhibited cell growth in 2D, and significantly inhibited mammosphere formation in multiple breast cancer cell lines. Importantly, ONC201 and TR-57 did not affect mitochondrial function, cell growth in 2D, nor mammosphere formation in CLPP KO cell lines, confirming ClpP is the target of these drugs. Flow cytometry analysis of MB231-SORE6-mCherry expressing cells demonstrated that ONC201 treatment significantly reduced the fraction of cells that were positive for the SORE6-mCherry activity, consistent with loss of CSCs. Finally, using in vivo limiting dilution experiments, CSC frequency was significantly lower (p<0.05) in the ONC201-treated group compared with the control group, indicating that ONC201 inhibits tumor initiation capability in breast cancers. Conclusion: Our studies suggest that targeting mitochondria by ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Voeller Donna, Raj Chari, Sam Gilbert, Binwu Tang, Christina Annunziata, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists inhibit cell growth and cancer stem cell function in breast cancer cells [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 4794.

A30 INHIBITION OF NF-KB SIGNALING IN DCLK1+ CELLS PROMOTES COLONIC INFLAMMATION AND COLITIS-ASSOCIATED CANCER

Abstract Background Colorectal cancer (CRC) is the 2nd leading cause of cancer death in Canada. A major risk factor for this disease is chronic inflammation. Despite the clear link between inflammation and cancer, the exact mechanism by which colitis leads to cancer is unknown. Our group has previously shown that a rare cell type in the gut marked by the expression of doublecortin-like kinase-1 (Dclk1) and known as a tuft cell, is quiescent, long-lived, and resistant to proliferation even upon mutation of the tumor suppressor APC. Interestingly, in the setting of colitis, these APC-mutated tuft cells become powerful cancer-initiating cells, but the mechanism by which this occurs is not known. NF-kB signaling is a major inflammatory pathway active in colitis and that has been linked to colorectal cancer. Inhibition of the NF-kB pathway in intestinal epithelial cells has also been shown to inhibit tumor initiation in a mouse model of colitis-associated cancer (Greten et al., 2004). Aims In the present study, we aim to examine the effect of NF-kB inhibition in tuft cells on colitis-associated cancer. Methods Dclk1CreERT2/APCf/f mice were crossed to IKK-β f/f mice and administered tamoxifen to conditionally knockout the function of both APC and IKK-β in tuft cells. Mice were then exposed to the colitis-inducing agent dextran sodium sulfate (DSS) to induce tumorigenesis. Approximately 16 weeks post-tamoxifen, colonic tumor number and size were analyzed to determine the effect of NF-kB pathway inhibition on tumor initiation and growth, respectively. Extent of inflammation was assessed by myeloperoxidase (MPO) activity and histological damage, and colonic tissue was collected for measurement of inflammatory mediators by qRT-PCR at both acute and long-term time points. Results Interestingly, at baseline we detected increased MPO activity in Dclk1CreERT2/APCf/f/IKK-β f/f mice compared to control mice, suggesting that inhibition of NF-kB in Dclk1+ cells may increase basal colonic inflammation. Consistent with this observation, inhibition of the NF-kB pathway also resulted in an increased number of tuft cell-derived tumors, with no observed change in tumor size. Acutely, we also observed an exacerbation of DSS-colitis in Dclk1CreERT2/APCf/f/IKK-β f/f mice, as detected by elevated MPO activity, increased histological damage, and reduced colon length compared to wildtype (IKK-β +/+) controls. Conclusions These data suggest that Dclk1+ cell-specific NF-kB signaling plays a key protective role against colitis and colitis-associated tumorigenesis. Targeting the NF-kB pathway may reduce the severity of colitis and the incidence of colitis-associated cancer. Funding Agencies CIHR

RRM2 Regulated By LINC00667/miR-143-3p Signal Is Responsible For Non-Small Cell Lung Cancer Cell Progression.

BackgroundNon-small cell lung cancer (NSCLC) is a common and fatal cancer worldwide with a very low 5-year overall survival rate. Ribonucleotide reductase M2 subunit (RRM2), a small subunit of the ribonucleotide reductase complex, has been found to be an oncogenic role in a variety of tumors including NSCLC. However, the regulatory mechanism of RRM2 in NSCLC is not clear. Increasing evidence suggests that non-coding RNAs (ncRNAs) including miRNAs and lincRNAs may promote or inhibit tumor initiation and development through regulating the expression of oncogenic genes. It is interesting to find ncRNAs which play important role in regulating RRM2 expression.Materials and methodsThe expression levels of RRM2, LINC0066 and miR-143-3p in NSCLC tumor tissues and cell lines were detected using qRT-PCR. The regulatory relationships among RRM2, LINC0066 and miR-143-3p were predicted using database analysis and verified by luciferase reporter assay and RIP analysis. The proliferation ability of NSCLC cells was assessed using CCK8 and colony formation assays. The expression of related proteins was determined by Western blot. In vivo effect of RRM2, LINC0066 and miR-143-3p to NSCLC were detected through xenograft experiments.ResultsIn this study, we found RRM2 was upregulated in NSCLC tumor and cell lines, and the aberrant upregulation predicted a poor prognosis. Then, we predicted and confirmed that RRM2 was negatively regulated by miR-143-3p. Further study implied that LINC00667 acted as a ceRNA by sponging miR-143-3p and regulated RRM2 expression indirectly. Moreover, we found that the growth of NSCLC was regulated by LINC00667/miR-143-3p/RRM2 signal pathway both in vitro and in vivo. LINC00667 and RRM2 promoted the tumor growth while miR-143-3p inhibited it.ConclusionOur study revealed a LINC00667/miR-143-3p/RRM2 signal pathway that played an important role in the progress of NSCLC, which might be potential therapeutic targets for NSCLC.

ADAM proteases: Emerging role and targeting of the non-catalytic domains.

ADAM proteases are multi domain transmembrane metalloproteases that cleave a range of cell surface proteins and activate signaling pathways implicated in tumor progression, including those mediated by Notch, EFGR, and the Eph receptors. Consequently, they have emerged as key therapeutic targets in the efforts to inhibit tumor initiation and progression. To that end, two main approaches have been taken to develop ADAM antagonists: (i) small molecule inhibitors, and (ii) monoclonal antibodies. In this mini-review we describe the distinct features of ADAM proteases, particularly of ADAM10 and ADAM17, their domain organization, conformational rearrangements, regulation, as well as their emerging importance as therapeutic targets in cancer. Further, we highlight an anti-ADAM10 monoclonal antibody that we have recently developed, which has shown significant promise in inhibiting Notch signaling and deterring growth of solid tumors in pre-clinical settings.

Elimination of KLK5 inhibits early skin tumorigenesis by reducing epidermal proteolysis and reinforcing epidermal microstructure

Epidermal desquamation involves a finely-tuned proteolytic cascade ensuring the regulated cleavage of desmosomes that releases old stratum corneum outermost layers. Although the roles of desmosomes in normal physiology are well-established, their putative involvement in cancer remains unexplored. The KLK5 protease is thought of having fundamental roles in epidermal proteolysis and homeostasis, and its aberrant activity has been linked to skin pathologies. We found that deletion of Klk5 results in significantly higher numbers of lengthier desmosomes and enhanced skin strength. Klk5−/− mice retained normal skin barrier function and are resistant to chemically-induced skin tumorigenesis. The resistance to tumorigenesis was not due to inhibition of inflammation, and on the contrary, absence of Klk5 increased the TPA-induced inflammatory skin response. We found that increased desmosomes and reduced proteolysis prevent oncogenic signaling by capturing β-catenin into the cytoplasm and facilitate epidermal keratinocyte apoptosis, thus, inhibiting tumor initiation. We highlight that the skin ultrastructure affects early neoplastic transformation by modulating intracellular signaling and suggest that tissue reinforcement provides a novel mode of tumor suppression.

Abstract 291: Synergistic inhibition of cancer invasion and metastasis by combined anti-PD1-TRC105-mediated Endoglin targeting on cancer-associated fibroblasts and endothelial cells

Abstract Cancer-associated fibroblasts (CAFs) are the major component of the tumor microenvironment in colorectal cancer (CRC) and play an important role in tumor progression and metastasis, partly through the transforming growth factor-β (TGF-β) signaling pathway. Recently, we showed that a subset of CAFs characterized by the expression of the TGF-β family co-receptor endoglin, selectively localized at the invasive borders of CRC tissues. Moreover, CAF-specific endoglin expression increased with higher tumor stage and predicted metastasis-free survival of stage-II CRC patients (n= 140, p<0.05). Targeting endoglin on CAFs using TRC105 (carotuximab), an endoglin neutralizing antibody currently in clinical development, inhibits endoglin dependent signaling and metastatic colonization when CRC cells and CAFs are co-injected into mice. We further investigated these anti-tumor effects in four different models of CRC (chemically-induced model of early CRC, subcutaneous MC38 and CT26 syngeneic mouse CRC models, and orthotopic MC38 model). TRC105 treatment inhibited tumor initiation (early stage CRC), growth, and progression. Therapeutic effects of TRC105 therapy were dependent on antibody dependent cytotoxicity (ADCC) and CD8+ T-cells, as no therapeutic benefit from TRC105 was observed in Fc-y I-IV receptor knockout mice or CD8+ cell depleted mice. Since therapeutic TRC105 activity was immune cell dependent, we combined TRC105 with anti-programmed cell death (PD-1 immunotherapy). We observed synergistic inhibition of tumor initiation and growth in all four tumor models, leading to delayed tumor outgrowth, increased survival and cure rates up to 60% in the combination treated mice bearing palpable tumors. Combined treatment also induced memory responses, as shown by resistance to repeated tumor challenge. Anti-tumor responses were accompanied by enhanced immune cell activation including an increased number of tumor-specific T-cells, NK cells, decreased number of tumor-infiltrating neutrophils and increased granzyme B production. CD8+ cytotoxic T-cells were instrumental in these synergistic therapeutic responses, as their depletion abolished the effects. Taken together, our data indicate significant therapeutic effects of targeting endoglin by affecting signaling and multiple immune related components in the tumor microenvironment. These results are currently being translated in a clinical trial combining TRC105 with nivolumab in patients with non-small cell lung cancer (NCT03181308). Citation Format: Mark JA Schoonderwoerd, Madelon Paauwe, Ricardo Angela, Maaike Koops, Tom Harryvan, Cornelis Sier, Marieke Fransen, Charles Theuer, Lukas JAC Hawinkels. Synergistic inhibition of cancer invasion and metastasis by combined anti-PD1-TRC105-mediated Endoglin targeting on cancer-associated fibroblasts and endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 291.

VEGF/Neuropilin Signaling in Cancer Stem Cells.

The function of vascular endothelial growth factor (VEGF) in cancer extends beyond angiogenesis and vascular permeability. Specifically, VEGF-mediated signaling occurs in tumor cells and this signaling contributes to key aspects of tumorigenesis including the self-renewal and survival of cancer stem cells (CSCs). In addition to VEGF receptor tyrosine kinases, the neuropilins (NRPs) are critical for mediating the effects of VEGF on CSCs, primarily because of their ability to impact the function of growth factor receptors and integrins. VEGF/NRP signaling can regulate the expression and function of key molecules that have been implicated in CSC function including Rho family guanosine triphosphatases (GTPases) and transcription factors. The VEGF/NRP signaling axis is a prime target for therapy because it can confer resistance to standard chemotherapy, which is ineffective against most CSCs. Indeed, several studies have shown that targeting either NRP1 or NRP2 can inhibit tumor initiation and decrease resistance to other therapies.