Expression Of Tumor-promoting Genes Research Articles

The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages

BackgroundTumor cells release extracellular vesicles (EVs) that contribute to the polarization of macrophages towards tumor-associated macrophages (TAMs). High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. However, there is a lack of understanding of whether IMP2 affects the cargo of cancer cell-derived EVs, thereby modulating macrophage polarization.MethodsEVs were isolated from IMP2-expressing HCT116 parental cells (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells. EVs were characterized according to MISEV guidelines, microRNA cargo was assessed by microRNA-Seq, and the protein cargo was analyzed by proteomics. Primary human monocyte-derived macrophages (HMDMs) were polarized by EVs, and the expression of genes and surface markers was assessed using qPCR and flow cytometry, respectively. Morphological changes of macrophages, as well as the migratory potential of cancer cells, were assessed by the Incucyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified using a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish larvae, and macrophages were isolated by fluorescence-activated cell sorting.ResultsEVs from WT and KO cells had a similar size and concentration and were positive for 25 vesicle markers. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs than KO EVs, while the expression of TNF and IL6 was reduced. A similar pattern was observed in macrophages from zebrafish larvae treated in vivo. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. MicroRNA-Seq revealed a significant difference in the microRNA composition of WT and KO EVs, particularly a high abundance of miR-181a-5p in WT EVs, which was absent in KO EVs. Inhibitors of macropinocytosis and phagocytosis antagonized the delivery of miR-181a-5p into macrophages and the downregulation of the miR-181a-5p target DUSP6. Proteomics data showed differences in protein cargo in KO vs. WT EVs, with the differentially abundant proteins mainly involved in metabolic pathways. WT EV-treated macrophages exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate than KO EV-treated cells.ConclusionOur results show that IMP2 determines the cargo of EVs released by cancer cells, thereby modulating the EVs' actions on macrophages. Expression of IMP2 is linked to the secretion of EVs that polarize macrophages towards a tumor-promoting phenotype.

Discovery of potent and novel dual NAMPT/BRD4 inhibitors for efficient treatment of hepatocellular carcinoma

The NAPRT-induced increase in NAD+ levels was proposed as a mechanism contributing to hepatocellular carcinoma (HCC) resistance to NAMPT inhibitors. Thus, concurrently targeting NAMPT and NAPRT could be considered to overcome drug resistance. A BRD4 inhibitor downregulates the expression of NAPRT in HCC, and the combination of NAMPT inhibitors with BRD4 inhibitors simultaneously blocks NAD+ generation via salvage and the PH synthesis pathway. Moreover, the combination of the two agents significantly downregulated the expression of tumor-promoting genes and strongly promoted apoptosis. The present work identified various NAMPT/BRD4 dual inhibitors based on the multitargeted drug rationale. Among them, compound A2, which demonstrated the strongest effect, exhibited potent inhibition of NAMPT and BRD4 (IC50 = 35 and 58 nM, respectively). It significantly suppressed the growth and migration of HCC cells and facilitated their apoptosis. Furthermore, compound A2 also manifested a robust anticancer effect in HCCLM3 xenograft mouse models, with no apparent toxic effects. Our findings in this study provide an effective approach to target NAD+ metabolism for HCC treatment.

Abstract 7266: VGLL4 is the target of the 3p25 homozygous deletion and presents a novel therapeutic vulnerability for TEAD1/4 but not TEAD1 inhibitors

Abstract The Hippo pathway, a critical cell proliferation regulator, remains an underexplored oncogenic pathway in precision oncology. It is executed through YAP1/TAZ co-activators and the TEAD transcription factor family (TEAD1-4), driving expression of tumor-promoting genes. Palmitic acid site TEAD inhibitors have shown strong pre-clinical and clinical activity, but identification of genetic markers of vulnerability and patient selection remain an urgent and open question. Sporos undertook a broad bioinformatic analysis of Hippo pathway components and regulatory genes to identify genetic alterations that drive TEAD activity and potentially act as targetable vulnerabilities. Here, we present a key finding from this investigation. The 3p25 locus undergoes frequent homozygous deletion in solid tumors especially lung squamous cell carcinoma. While VHL was long assumed to be the target tumor suppressor gene of this deletion - we show that VGLL4 is in the only gene in the peak of GISTIC statistically significance in lung squamous cell carcinoma and VGLL4 and ATG7 are both in the peak in Renal Clear Cell Carcinoma (RCC). These data unambiguously establish that VGLL4 deletion is a major oncogenic driver in lung squamous cell carcinoma (SCC) and a contributor in RCC. Because VGLL4 is the major negative relator of the YAP/TEAD transcriptional complex and directly competes with YAP for TEAD binding - we hypothesize that Lung SCC with VGLL4-homozygous deletion would be uniquely dependent on YAP/TEAD activity and show selective sensitivity to TEAD inhibitors. While we were unable to locate a Lung SCC VGLL4-deleted PDX, we identified a VGLL4/ATG7 homozygous deleted RCC PDX, KI2552, in the CrownBio collection. We show that treatment with SPR1, Sporos’s novel TEAD1/4 inhibitor, decreases tumor growth and dramatically extends survival of mice bearing VGLL4/ATG7-deleted KI2552 RCC PDX. On the other hand, VT103, a strong TEAD1 inhibitor which shows comparable pre-clinical activity to SPR1 in the NCI-H226 mesothelioma CDX - did not show any activity in KI2552 PDX. These data emphasize the importance of targeting paralogs besides TEAD1 to broaden anti-neoplastic activity beyond mesothelioma and suggest that VGLL4-homozygous deletion confers TEAD inhibitor sensitivity even in carcinomas where it isn’t the principal driver and portent exceptionally anti-neoplastic activity in neoplasms where it is. Given the wide distribution of the 3p25 homozygous deletion - our findings open a large new potential responder population to TEAD inhibitors and warrant inclusion of VGLL4 in NSG diagnostic sequencing panels. Citation Format: Florian Muller, Selvi Kunnimalaiyaan, Parth Mangrolia, Jill Olson. VGLL4 is the target of the 3p25 homozygous deletion and presents a novel therapeutic vulnerability for TEAD1/4 but not TEAD1 inhibitors [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 7266.

Optimizing aspirin dose for colorectal cancer patients through deep phenotyping using novel biomarkers of drug action.

Background: Low-dose aspirin's mechanism of action for preventing colorectal cancer (CRC) is still debated, and the optimal dose remains uncertain. We aimed to optimize the aspirin dose for cancer prevention in CRC patients through deep phenotyping using innovative biomarkers for aspirin's action. Methods: We conducted a Phase II, open-label clinical trial in 34 CRC patients of both sexes randomized to receive enteric-coated aspirin 100mg/d, 100mg/BID, or 300mg/d for 3 ± 1weeks. Biomarkers were evaluated in blood, urine, and colorectal biopsies at baseline and after dosing with aspirin. Novel biomarkers of aspirin action were assessed in platelets and colorectal tissues using liquid chromatography-mass spectrometry to quantify the extent of cyclooxygenase (COX)-1 and COX-2 acetylation at Serine 529 and Serine 516, respectively. Results: All aspirin doses caused comparable % acetylation of platelet COX-1 at Serine 529 associated with similar profound inhibition of platelet-dependent thromboxane (TX)A2 generation ex vivo (serum TXB2) and in vivo (urinary TXM). TXB2 was significantly reduced in CRC tissue by aspirin 300mg/d and 100mg/BID, associated with comparable % acetylation of COX-1. Differently, 100mg/day showed a lower % acetylation of COX-1 in CRC tissue and no significant reduction of TXB2. Prostaglandin (PG)E2 biosynthesis in colorectal tumors and in vivo (urinary PGEM) remained unaffected by any dose of aspirin associated with the variable and low extent of COX-2 acetylation at Serine 516 in tumor tissue. Increased expression of tumor-promoting genes like VIM (vimentin) and TWIST1 (Twist Family BHLH Transcription Factor 1) vs. baseline was detected with 100mg/d of aspirin but not with the other two higher doses. Conclusion: In CRC patients, aspirin 300mg/d or 100mg/BID had comparable antiplatelet effects to aspirin 100mg/d, indicating similar inhibition of the platelet's contribution to cancer. However, aspirin 300mg/d and 100mg/BID can have additional anticancer effects by inhibiting cancerous tissue's TXA2 biosynthesis associated with a restraining impact on tumor-promoting gene expression. EUDRACT number: 2018-002101-65. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT03957902.

An unappreciated cell survival-independent role for BAFF initiating chronic lymphocytic leukemia.

Chronic Lymphocytic Leukemia (CLL) is characterized by the expansion of CD19+ CD5+ B cells but its origin remains debated. Mutated CLL may originate from post-germinal center B cells and unmutated CLL from CD5+ mature B cell precursors. Irrespective of precursor types, events initiating CLL remain unknown. The cytokines BAFF and APRIL each play a significant role in CLL cell survival and accumulation, but their involvement in disease initiation remains unclear. We generated novel CLL models lacking BAFF or APRIL. In vivo experiments were conducted to explore the impact of BAFF or APRIL loss on leukemia initiation, progression, and dissemination. Additionally, RNA-seq and quantitative real-time PCR were performed to unveil the transcriptomic signature influenced by BAFF in CLL. The direct role of BAFF in controlling the expression of tumor-promoting genes was further assessed in patient-derived primary CLL cells ex-vivo. Our findings demonstrate a crucial role for BAFF, but not APRIL, in the initiation and dissemination of CLL cells. In the absence of BAFF or its receptor BAFF-R, the TCL1 transgene only increases CLL cell numbers in the peritoneal cavity, without dissemination into the periphery. While BAFF binding to BAFF-R is dispensable for peritoneal CLL cell survival, it is necessary to activate a tumor-promoting gene program, potentially linked to CLL initiation and progression. This direct role of BAFF in controlling the expression of tumor-promoting genes was confirmed in patient-derived primary CLL cells ex-vivo. Our study, involving both mouse and human CLL cells, suggests that BAFF might initiate CLL through mechanisms independent of cell survival. Combining current CLL therapies with BAFF inhibition could offer a dual benefit by reducing peripheral tumor burden and suppressing transformed CLL cell output.

NK3.3-Derived Extracellular Vesicles Penetrate and Selectively Kill Treatment-Resistant Tumor Cells.

Cancer treatments often become ineffective due to the development of tumor resistance, leading to metastasis and relapse. Treatments may also fail because of their inability to access cells deep within the tumor tissue. When this occurs, new therapeutic agents are needed. We previously reported that NK3.3EVs, extracellular vesicles (EVs) derived from the normal human natural killer (NK) cell line, NK3.3, have strong cytotoxic activity against leukemia and breast cancer cell lines, without harming normal cells. Here, we used a three-dimensional (3D) MCF7 breast cancer mammosphere model to reproduce a more physiological environment that NK3.3EVs would encounter in vivo. NK3.3EVs penetrated MCF7 mammospheres, inducing death by apoptosis. We generated an imatinib-resistant K562 chronic myeloid leukemia (CML) cell line to investigate whether NK3.3EVs were able to kill tumor cells resistant to front-line chemotherapy. NK3.3EVs were even more cytotoxic to imatinib-resistant cells than parental cells, inducing apoptosis via caspase-3/-7 activation. The small population of cancer stem cells (CSCs) within tumors also contributes to therapeutic resistance. NK3.3EVs reduced the CSC-like CD34+/CD38- subpopulation in imatinib-resistant and parental K562 cultures and decreased CSC-associated expression of tumor-promoting genes. Our results provide strong evidence that NK3.3EVs may be a potential new immunotherapeutic agent for difficult-to-treat cancers.

Abstract 6776: Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC

Abstract Background: While cancer therapies are becoming increasingly more targeted, our understanding of the genomic changes occurring during these treatments is lacking. Moreover, the tumoral heterogeneity and its spatial distribution remain important obstacles in predicting tumor evolution and treatment responses. In recent years, genomic spatial profiling techniques have shed light on this complexity by detecting gene expression changes in heterogenous tumor regions and their microenvironment over the course of treatment. Methods: Tepotinib is a highly selective and potent MET class Ib inhibitor, approved for the treatment of patients with NSCLC with MET exon 14 skipping alterations. To detect genomic changes during neoadjuvant treatment with tepotinib, a GeoMx® digital spatial profiler (DSP; Nanostring Technologies) analysis was conducted in paired baseline and on-treatment patient samples. GeoMx® DSP was performed by selecting regions of interest with nuclear, TTF-1, CD3 and CD33 morphology markers followed by next-generation sequencing (NGS) using the Cancer Transcriptome Atlas RNA panel (>1,800 cancer-related genes). Results: Following a poor response to chemotherapy, neoadjuvant tepotinib treatment was initiated in a 54-year-old female, never-smoker with NSCLC harboring a MET exon 14 alteration. Owing to a good response to tepotinib, surgical resection of the tumor was decided by the tumor board given the downgrading (from IIIB to IA1) and resulting in a major pathological response. This resection allowed spatial transcriptomics to be compared to a baseline biopsy from the same patient. MET signaling inhibition by tepotinib treatment increased immune cell infiltration in the tumor microenvironment compared to the baseline sample. This enhanced tumoral immune cell density was shown by the fluorescent morphology markers and confirmed through spatial deconvolution of the NGS results. It was further characterized by the significantly altered gene expression levels between the two samples. Gene ontology (GO) assessment of the differentially expressed genes revealed GO terms associated with tumorigenesis in the basal sample, such as cell mobility and motility, vascular epithelial growth factor production and cell communication. In turn, the tepotinib-treated tissue mainly exhibited tumor-clearing characteristics, including antigen processing and presentation through major histocompatibility complex classes I & II, T cell-mediated toxicity, and granulocyte and leukocyte activation. Conclusion: This is the first spatial genomic characterization of the effect of tepotinib on the tumor and its microenvironment in a patient with NSCLC over time. Results from this case report highlight the impact of MET inhibition by tepotinib on the tumor immune microenvironment, likely enabling the recruitment of antigen-presenting cells for tumor clearance, while reducing the expression of tumor-promoting genes. Citation Format: Manon A. Simard, Felix Geist, Carlos Cabrera, Santiago Viteri, Joachim Albers, Michael Zuehlsdorf, Niki Karachaliou. Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6776.

Abstract P005: SLC38A5 characterization and its tumor promoting role in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal and aggressive cancers. Based on the Human Protein Atlas database, solute carrier 38A5 (SLC38A5) is significantly upregulated in PDAC and correlates with poor patient survival. SLC38A5 is a Na+-coupled, electroneutral amino acid transporter that transports glutamine, serine, glycine, and methionine that are essential for glutaminolysis and one-carbon metabolism pathways on which cancer cells are dependent. Furthermore, SLC38A5 is a c-Myc target and induces macropinocytosis. Thus, these attributes of SLC38A5 in cancer cells makes it a favorable contribution to their tumor-promoting gene expression programs. Using real-time PCR and Western blotting, we validated the expression status of SLC38A5 in PDAC cell lines, patient derived xenografts (PDX), and organoids. Using radiolabeled serine uptake, we also confirmed that PDAC cell lines express functional SLC38A5. To investigate the tumor promoting role of SLC38A5, shRNA-mediated knockdown of SLC38A5 was performed in BxPC-3 and HPAF-II human PDAC cell lines. After validating SLC38A5 knockdown using real-time PCR and radiolabeled serine uptake, colony formation and transwell invasion assay were performed using the knockdown cells and non-targeting controls (NTC). Interestingly, SLC38A5 knockdown suppressed the colony formation ability as well as the invasion capacity in both cell lines, suggesting the tumor promoting role of SLC38A5. To extrapolate the in vitro data further, we performed subcutaneous xenograft using SLC38A5-silenced BxPC-3 and HPAF-II cells into the athymic nude mice. Interestingly, we found that SLC38A5 knockdown cells grew slower, as evidenced by smaller tumor weight, further validating our in vitro data. Our future work involves extrapolating these data in the KPC (Kras, p53, and PDX-1-Cre) spontaneous mouse model of PDAC and elucidating its mechanism of upregulation. Citation Format: Tyler J. Sniegowski, Vadivel Ganapathy1, Yangzom Bhutia. SLC38A5 characterization and its tumor promoting role in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P005.

EVI1 activates tumor-promoting transcriptional enhancers in pancreatic cancer.

Cancer cells utilize epigenetic alterations to acquire autonomous capabilities for tumor maintenance. Here, we show that pancreatic ductal adenocarcinoma (PDA) cells utilize super-enhancers (SEs) to activate the transcription factor EVI1 (ecotropic viral integration site 1) gene, resulting in activation of an EVI1-dependent transcription program conferring PDA tumorigenesis. Our data indicate that SE is the vital cis-acting element to maintain aberrant EVI1 transcription in PDA cells. Consistent with disease progression and inferior survival outcomes of PDA patients, we further show that EVI1 upregulation is a major cause of aggressive tumor phenotypes. Specifically, EVI1 promotes anchorage-independent growth and motility in vitro and enhances tumor propagation in vivo. Mechanistically, EVI1-dependent activation of tumor-promoting gene expression programs through the stepwise configuration of the active enhancer chromatin attributes to these phenotypes. In sum, our findings support the premise that EVI1 is a crucial driver of oncogenic transcription programs in PDA cells. Further, we emphasize the instructive role of epigenetic aberrancy in establishing PDA tumorigenesis.

Nanotherapy delivery of c-myc inhibitor targets Protumor Macrophages and preserves Antitumor Macrophages in Breast Cancer.

Tumor-associated macrophages (TAMs) enhance tumor growth in mice and are correlated with a worse prognosis for breast cancer patients. While early therapies sought to deplete all macrophages, current therapeutics aim to reprogram pro-tumor macrophages (M2) and preserve those necessary for anti-tumor immune responses (M1). Recent studies have shown that c-MYC (MYC) is induced in M2 macrophages in vitro and in vivo where it regulates the expression of tumor-promoting genes. In a myeloid lineage MYC KO mouse model, MYC had important roles in macrophage maturation and function leading to reduced tumor growth. We therefore hypothesized that targeted delivery of a MYC inhibitor to established M2 TAMs could reduce polarization toward an M2 phenotype in breast cancer models.Methods: In this study, we developed a MYC inhibitor prodrug (MI3-PD) for encapsulation within perfluorocarbon nanoparticles, which can deliver drugs directly to the cytosol of the target cell through a phagocytosis independent mechanism. We have previously shown that M2-like TAMs express significant levels of the vitronectin receptor, integrin β3, and in vivo targeting and therapeutic potential was evaluated using αvβ3 integrin targeted rhodamine-labeled nanoparticles (NP) or integrin αvβ3-MI3-PD nanoparticles.Results: We observed that rhodamine, delivered by αvβ3-rhodamine NP, was incorporated into M2 tumor promoting macrophages through both phagocytosis-independent and dependent mechanisms, while NP uptake in tumor suppressing M1 macrophages was almost exclusively through phagocytosis. In a mouse model of breast cancer (4T1-GFP-FL), M2-like TAMs were significantly reduced with αvβ3-MI3-PD NP treatment. To validate this effect was independent of drug delivery to tumor cells and was specific to the MYC inhibitor, mice with integrin β3 knock out tumors (PyMT-Bo1 β3KO) were treated with αvβ3-NP or αvβ3-MI3-PD NP. M2 macrophages were significantly reduced with αvβ3-MI3-PD nanoparticle therapy but not αvβ3-NP treatment.Conclusion: These data suggest αvβ3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.

CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factorstimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness.

The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies.

Mutant p53 regulates enhancer-associated H3K4 monomethylation through interactions with the methyltransferase MLL4

Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary in vitro studies with recombinant chromatin and purified proteins demonstrate that binding of the MLL3/4 complex and H3K4me1 deposition is enhanced by mutant p53 and p300-mediated acetylation, which in turn reflects a MLL3/4-dependent enhancement of mutant p53 and p300-dependent transcriptional activation. Collectively, our findings establish a mechanism in which mutant p53 cooperates with MLL4 to regulate aberrant enhancer activity and tumor-promoting gene expression in response to chronic immune signaling.

Salivary Gland Cancer Patient-Derived Xenografts Enable Characterization of Cancer Stem Cells and New Gene Events Associated with Tumor Progression.

Purpose: Salivary gland cancers (SGC) frequently present with distant metastases many years after diagnosis, suggesting a cancer stem cell (CSC) subpopulation that initiates late recurrences; however, current models are limited both in their availability and suitability to characterize these rare cells.Experimental Design: Patient-derived xenografts (PDX) were generated by engrafting patient tissue onto nude mice from one acinic cell carcinoma (AciCC), four adenoid cystic carcinoma (ACC), and three mucoepidermoid carcinoma (MEC) cases, which were derived from successive relapses from the same MEC patient. Patient and PDX samples were analyzed by RNA-seq and Exome-seq. Sphere formation potential and in vivo tumorigenicity was assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.Results: For successive MEC relapses we found a time-dependent increase in CSCs (ALDH+CD44high), increasing from 0.2% to 4.5% (P=0.033), but more importantly we observed an increase in individual CSC sphere formation and tumorigenic potential. A 50% increase in mutational burden was documented in subsequent MEC tumors, and this was associated with increased expression of tumor-promoting genes (MT1E, LGR5, and LEF1), decreased expression of tumor-suppressor genes (CDKN2B, SIK1, and TP53), and higher expression of CSC-related proteins such as SOX2, MYC, and ALDH1A1. Finally, genomic analyses identified a novel NFIB-MTFR2 fusion in an ACC tumor and confirmed previously reported fusions (NTRK3-ETV6 and MYB-NFIB)Conclusions: Sequential MEC PDX models preserved key patient features and enabled the identification of genetic events putatively contributing to increases in both CSC proportion and intrinsic tumorigenicity, which mirrored the patient's clinical course. Clin Cancer Res; 24(12); 2935-43. ©2018 AACR.

Therapeutic targeting of tumor-associated macrophages through microscale engineering of the prostate cancer microenvironment.

184 Background: The tumor microenvironment (TME) plays integral roles in prostate cancer progression and is a high value therapeutic target. The development of effective TME-targeted therapies is limited by current technologies which are insufficient to replicate or analyze this complex environment. To address these challenges, we have developed a microfluidic cell culture platform known as STACKS, which permits co-culture of up to 6 patient-derived cell populations as well as compartmentalized, multiplexed analysis of gene expression, cell signaling, and matrix remodeling. We have focused on investigation of tumor-associated macrophages (TAMs), which have been implicated in prostate cancer growth, metastasis, survival, and therapeutic resistance. Methods: Using blood and prostate tissue obtained through biomarker studies at the University of Wisconsin, we isolated patient-derived cell populations for culture and analysis within the STACKS device. Monocytes obtained from peripheral blood were differentiated into tumor inhibitory (M1) and tumor promoting (M2) macrophages in the device. RNA, protein, and secretory factor expression were analyzed following manipulations of culture surface, cytokine exposure, and multi-cell culture with tumor and stromal cell populations. Results: We report that physical properties of the TME, such as topography and hydrophilicity, impact expression of tumor-promoting genes within macrophages. We also characterized macrophage expression of pro- and antitumor genes, including CCL18, CXCL10, CXCL11, IL-10, IL-12b, VEGF, and identified expression profiles that are dependent on tumor and stromal cell populations in co-culture and diverge from classical M1/M2 characterization. Conclusions: The STACKS platform is capable of advanced modeling and analysis of the prostate cancer TME. Investigation of patient-derived monocytes within STACKS has identified physical properties of the TME that affect tumor-related functions of macrophages and has also demonstrated that an M1/M2 classification spectrum may not be sufficient for characterizing TAMs. These findings represent new therapeutic targets for prostate cancer treatment.

Abstract 1693: Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases

Abstract Background/Purpose: Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the biotransformation of many drugs and xenobiotic compounds. It is overexpressed in different cancers and has been shown to enhance the migratory and invasive activity of cancer cells. Additionally, NNMT is an important regulator of the methylation potential in cells by consumption of S-adenosyl methionine (SAM), an important substrate for methyltransferases. By this mechanism NNMT reduces methylation of proteins such as certain histones leading to epigenetic remodeling and induced expression of tumor-promoting genes (Ulanovskaya et al, Nat. Chem. Biol. 2013). In clear cell renal cell carcinoma (ccRCC), the dominant histological subtype of renal cell cancer, NNMT is expressed at high levels compared to normal kidney tissue and could represent a novel therapeutic target, especially for the treatment of metastatic disease which is marked by a five-year survival rate of below 10% with currently available therapies. Methods: We analyzed NNMT in a ccRCC patient cohort from The Cancer Genome Atlas (TCGA) concerning mRNA expression (n = 463), somatic variations (n = 410) and association with clinical parameters and patient survival. In an independent collection of ccRCC tissues (n = 64), corresponding normal kidney tissues and metastatic tissues from different organs (n = 129), NNMT mRNA and protein expression were quantified via Real-Time PCR and immunohistochemical staining of tissue microarrays, respectively. Results: In both cohorts, NNMT mRNA expression was significantly upregulated in tumor compared to non-tumor tissue. Somatic mutations and copy number alterations in the NNMT gene locus were observed in only a small number of patients. NNMT mRNA expression correlated weakly with tumor stage and grading in both cohorts, but not with the presence of lymph node or distant metastases. In Kaplan-Meier analyses high NNMT expression was associated with worse overall and cancer-specific survival. In ccRCC metastases from different organ sites, NNMT mRNA and protein expression were also induced compared to normal kidney tissue and high expression was again associated with worse patient survival in Kaplan-Meier analysis (HR = 2.15, Plog-rank = 0.05). Conclusion: NNMT mRNA and protein are highly expressed not only in primary tissue of ccRCC, but also in metastases derived from different organs, offering it as a potential drug-target for metastatic ccRCC. Although NNMT expression is a good prognostic indicator for patient survival, its molecular function in ccRCC tumorigenesis and metastasis formation has to be further evaluated. Supported by the Robert Bosch Foundation, Stuttgart, Germany and the ICEPHA Grant Tuebingen-Stuttgart, Germany Citation Format: Anna Reustle, Steffen Rausch, Stefan Winter, Florian Büttner, Stephan Kruck, Joerg Hennenlotter, Marcus Scharpf, Falko Fend, Jens Bedke, Arnulf Stenzl, Matthias Schwab, Elke Schaeffeler. Nicotinamide N-methyltransferase in clear cell renal cell carcinoma primary tumors and metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1693.

Chapter Five - ATP-Dependent Chromatin Remodeling Complexes as Novel Targets for Cancer Therapy

The progression to advanced stage cancer requires changes in many characteristics of a cell. These changes are usually initiated through spontaneous mutation. As a result of these mutations, gene expression is almost invariably altered allowing the cell to acquire tumor-promoting characteristics. These abnormal gene expression patterns are in part enabled by the posttranslational modification and remodeling of nucleosomes in chromatin. These chromatin modifications are established by a functionally diverse family of enzymes including histone and DNA-modifying complexes, histone deposition pathways, and chromatin remodeling complexes. Because the modifications these enzymes deposit are essential for maintaining tumor-promoting gene expression, they have recently attracted much interest as novel therapeutic targets. One class of enzyme that has not generated much interest is the chromatin remodeling complexes. In this review, we will present evidence from the literature that these enzymes have both causal and enabling roles in the transition to advanced stage cancers; as such, they should be seriously considered as high-value therapeutic targets. Previously published strategies for discovering small molecule regulators to these complexes are described. We close with thoughts on future research, the field should perform to further develop this potentially novel class of therapeutic target.

Regulation of membrane-type 1 matrix metalloproteinase expression by zonula occludens-2 in human lung cancer cells

During tumor invasion, tumor epithelial cells acquire migratory and invasive properties involving important phenotypic alterations. Among these changes, one can observe reorganization or a loss of cell-cell adhesion complexes such as tight junctions (TJs). TJs are composed of transmembrane proteins (occludin, claudins) linked to the actin cytoskeleton through cytoplasmic adaptor molecules including those of the zonula occludens family (ZO-1, -2, -3). We here evaluated the potential role of ZO-2 in the acquisition of invasive properties by tumor cells. In vivo, we showed a decrease of ZO-2 expression in bronchopulmonary cancers, with a preferential localization in the cytoplasm. In addition, in vitro, the localization of ZO-2 varied according to invasive properties of tumor cells, with a cytoplasmic localization correlating with invasion. In addition, we demonstrated that ZO-2 inhibition increases invasive and migrative capacities of invasive tumor cells. This was associated with an increase of MT1-MMP. These results suggest that ZO-2, besides its structural role in tight junction assembly, can act also as a repressor of tumor progression through its ability to reduce the expression of tumor-promoting genes in invasive tumor cells.

Selenium-binding protein 1: Its physiological function, dependence on aryl hydrocarbon receptors, and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin

BackgroundSelenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1. MethodsTo address this issue, we generated Selenbp1-null [Selenbp1 (−/−)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model. ResultsSelenbp1 (−/−) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (−/−) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (−/−) and (+/+) mice. ConclusionsThe above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity. General significanceSelenbp1 (−/−) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.

Role of Urokinase Receptor in Tumor Progression and Development

Elevated level of urokinase receptor (uPAR) is detected in various aggressive cancer types and is closely associated with poor prognosis of cancers. Binding of uPA to uPAR triggers the conversion of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. uPA-uPAR interaction also elicits signals that stimulate cell proliferation/survival and the expression of tumor-promoting genes, thus assisting tumor development. In addition to its interaction with uPA, uPAR also interacts with vitronectin and this interaction promotes cancer metastasis by activating Rac and stimulating cell migration. Although underlying mechanisms are yet to be fully elucidated, uPAR has been shown to facilitate epithelial-mesenchymal transition (EMT) and induce cancer stem cell-like properties in breast cancer cells. The fact that uPAR lacks intracellular domain suggests that its signaling must be mediated through its co-receptors. Indeed, uPAR interacts with diverse transmembrane proteins including integrins, ENDO180, G protein-coupled receptors and growth factor receptors in cancer cells and these interactions are proven to be critical for the role of uPAR in tumorigenesis. Inhibitory peptide that prevents uPA-uPAR interaction has shown the promise to prolong patients' survival in the early stage of clinical trial. The importance of uPAR's co-receptor in uPAR's tumor-promoting effects implicate that anti-cancer therapeutic agents may also be developed by disrupting the interactions between uPAR and its functional partners.