Tumor Expression Levels Research Articles

Transcriptomics in Tumor and Normal Lung Tissues Identify Patients With Early-Stage Non-Small-Cell Lung Cancer With High Risk of Postsurgery Recurrence Who May Benefit From Adjuvant Therapies.

PURPOSEThe prognosis of patients with non–small-cell lung cancer (NSCLC), traditionally determined by anatomic histology and TNM staging, neglects the biological features of the tumor that may be important in determining patient outcome and guiding therapeutic interventions. Identifying patients with NSCLC at increased risk of recurrence after curative-intent surgery remains an important unmet need so that known effective adjuvant treatments can be offered to those at highest risk of recurrence.METHODSRelative gene expression level in the primary tumor and normal bronchial tissues was used to retrospectively assess their association with disease-free survival (DFS) in a cohort of 120 patients with NSCLC who underwent curative-intent surgery.RESULTSLow versus high Digital Display Precision Predictor (DDPP) score (a measure of relative gene expression) was significantly associated with shorter DFS (highest recurrence risk; P = .006) in all patients and in patients with TNM stages 1-2 (P = .00051; n = 83). For patients with stages 1-2 and low DDPP score (n = 29), adjuvant chemotherapy was associated with improved DFS (P = .0041). High co-overexpression of CTLA-4, PD-L1, and ICOS in normal lung (28 of 120 patients) was also significantly associated with decreased DFS (P = .0013), suggesting an immune tolerance to tumor neoantigens in some patients. Patients with DDPP low and immunotolerant normal tissue had the shortest DFS (P = 2.12E–11).CONCLUSIONTNM stage, DDPP score, and immune competence status of normal lung are independent prognostic factors in multivariate analysis. Our findings open new avenues for prospective prognostic assessment and treatment assignment on the basis of transcriptomic profiling of tumor and normal lung tissue in patients with NSCLC.

Evaluation of the expression pattern and diagnostic value of PPARγ in malignant and benign primary bone tumors

PurposeThe quantifiable description of PPARγ expression pattern beside mechanistic in-vitro evidence will provide insights into the involvement of this mediator in tumor pathogenesis. This study is focused on illuminating the PPARγ gene and protein expression pattern, its association with tumor deterioration and its diagnostic value in different types of primary bone tumors.MethodsThe expression pattern of PPARγ was investigated in the 180 bone tissues including 90 bone tumor tissues and 90 non-cancerous bone tissues. The local PPARγ expression level was assessed using real-time qRT-PCR and the PPARγ protein expression pattern was measured using immunohistochemistry. The correlation of PPARγ expression level with patients’ clinic-pathological features, also the value of the variables in predicting PPARγ expression level in tumors and the value of PPARγ to discriminate tumor subtypes were assessed.ResultsThe mean PPARγ mRNA expression was significantly higher in bone tumors compared to healthy bone tissues, also the malignant tumors including osteosarcoma and Ewing sarcoma had the elevated level of PPARγ mRNA compared to GCT tumors. Consistently, the protein expression of PPARγ in the tumor site was significantly higher in the bone tumors and malignant tumors compared to non-cancerous and benign tumors, respectively. The PPARγ protein could predict malignant tumor features including tumor grade, metastasis and recurrence significantly. Moreover, PPARγ could potentially discriminate the patients from the controls also malignant tumors from benign tumors with significant sensitivity and specificity.ConclusionsPPARγ might be involved in primary bone tumor pathogenesis and determining its molecular mechanism regarding bone cancer pathogenesis is of grave importance.

High expression of nuclear NRF2 combined with NFE2L2 alterations predicts poor prognosis in esophageal squamous cell carcinoma patients

AbstractNuclear factor erythroid-2 related factor-2 (NFE2L2 or NRF2) is a frequently mutated gene in esophageal squamous cell carcinoma (ESCC). However, the roles of NFE2L2 alterations in ESCC remain elusive. In order to elucidate this issue, 130 ESCC patients who underwent esophagectomy were enrolled. The majority of tumor tissues were positive for NRF2, which was significantly enriched in the nucleus of the primary tumor tissues compared with the noncancerous mucosae. Primary ESCC tumors positive for NRF2 tended to be positive for NAD(P)H quinone oxidoreductase 1 (NQO1) as the downstream target of NRF2. There was a positive correlation between NRF2 and NQO1 expression level in primary tumors. NQO1 staining in primary tumors with NRF2 nuclear expression was significantly stronger than that with NRF2 cytoplasmic expression. In addition, high concordance for the status of NRF2 expression between primary tumors and corresponding metastatic lesions was observed. Next, we found high expression of nuclear NRF2 (the proportion of nuclear NRF2 expression >20% or nuclear NRF2 immunohistochemistry score >20) predicted shorter overall survival in patients with dual-positive expression of NRF2 and NQO1. Captured-based targeted sequencing revealed that NFE2L2 somatic alterations were observed in 52.8% of ESCC patients with dual-positive expression of NRF2 and NQO1. NFE2L2 amplification and mutations within the DLG/ETGE motifs were seen more frequently in ESCC tumors with nuclear or nucleocytoplasmic expression of NRF2 compared with those with cytoplasmic expression of NRF2. We also found high expression of nuclear NRF2 plus the status of NFE2L2 alteration exhibited high performance in predicting prognosis of ESCC patients. Our study demonstrated that high nuclear NRF2 expression and NFE2L2 alterations were associated with poor prognosis of ESCC patients. These findings suggest that NRF2 signaling pathway might play vital roles in ESCC malignancy and the aberrant activation of NRF2 pathway predicts unfavorable prognosis in ESCC.

Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis

ABSTRACT Macroautophagy/autophagy is an evolutionarily conserved cellular stress response mechanism. Autophagy induction in the tumor microenvironment (stroma) has been shown to support tumor metabolism. However, cancer cell-derived secreted factors that initiate communication with surrounding cells and stimulate autophagy in the tumor microenvironment are not fully documented. We identified CTF1/CT-1 (cardiotrophin 1) as an activator of autophagy in fibroblasts and breast cancer-derived carcinoma-associated fibroblasts (CAFs). We showed that CTF1 stimulated phosphorylation and nuclear translocation of STAT3, initiating transcriptional activation of key autophagy proteins. Additionally, following CTF1 treatment, AMPK and ULK1 activation was observed. We provided evidence that autophagy was important for CTF1-dependent ACTA2/α-SMA accumulation, stress fiber formation and fibroblast activation. Moreover, promotion of breast cancer cell migration and invasion by activated fibroblasts depended on CTF1 and autophagy. Analysis of the expression levels of CTF1 in patient-derived breast cancer samples led us to establish a correlation between CTF1 expression and autophagy in the tumor stroma. In line with our in vitro data on cancer migration and invasion, higher levels of CTF1 expression in breast tumors was significantly associated with lymph node metastasis in patients. Therefore, CTF1 is an important mediator of tumor-stroma interactions, fibroblast activation and cancer metastasis, and autophagy plays a key role in all these cancer-related events. Abbreviations: ACTA2/α-SMA: actin, alpha 2, smooth muscle CAFs: cancer- or carcinoma-associated fibroblasts CNT Ab.: control antibody CNTF: ciliary neurotrophic factor CTF1: cardiotrophin 1 CTF1 Neut. Ab.: CTF1-specific neutralizing antibody GFP-LC3 MEF: GFP-fused to MAP1LC3 protein transgenic MEF LIF: leukemia inhibitory factor IL6: interleukin 6 MEFs: mouse embryonic fibroblasts MEF-WT: wild-type MEFs OSM: oncostatin M TGFB/TGFβ: transforming growth factor beta

Research Progress of Long Non-Coding RNA GAS5 in Malignant Tumors.

With completing the whole genome sequencing project, awareness of lncRNA further deepened. The growth arrest-specific transcript 5 (GAS5) was initially identified in growth-inhibiting cells. GAS5 is a lncRNA (long non-coding RNA), and it plays a crucial role in various human cancers. There are small ORFs (open reading frames) in the exons of the GAS5 gene sequence, but they do not encode functional proteins. In addition, GAS5 is also the host gene of several small nucleolar RNAs (snoRNA). These snoRNAs are believed to play a suppressive role during tumor progression by methylating ribosomal RNA (rRNA). As a result, GAS5 expression levels in tumor tissues are significantly reduced, leading to increased malignancy, poor prognosis, and drug resistance. Recent studies have demonstrated that GAS5 can interact with miRNAs by base-pairing and other functional proteins to inhibit their biological functions, impacting signaling pathways and changing the level of intracellular autophagy, oxidative stress, and immune cell function in vivo. In addition, GAS5 participates in regulating proliferation, invasion, and apoptosis through the above molecular mechanisms. This article reviews the recent discoveries on GAS5, including its expression levels in different tumors, its biological behavior, and its molecular regulation mechanism in human cancers. The value of GAS5 as a molecular marker in the prevention and treatment of cancers is also discussed.

Validation of a Patient-Derived Xenograft Model for Cervical Cancer Based on Genomic and Phenotypic Characterization.

Simple SummaryThe rate of total tumor engraftment of patient-derived xenografts is 50% in cervical cancer. These cancers retain their histopathological characteristics. The gene mutations and expression patterns associated with carcinogenesis and infiltration and the expression levels of genes in extracellular vesicles released from the tumors are similar between patient-derived xenograft models and primary tumors. Patient-derived xenograft models of cervical cancer could be potentially useful tools for translational research.Patient-derived xenograft (PDX) models are useful tools for preclinical drug evaluation, biomarker identification, and personalized medicine strategies, and can be developed by the heterotopic or orthotopic grafting of surgically resected tumors into immunodeficient mice. We report the PDX models of cervical cancer and demonstrate the similarities among original and different generations of PDX tumors. Fresh tumor tissues collected from 22 patients with primary cervical cancer were engrafted subcutaneously into NOD.CB17-PrkdcSCID/J mice. Histological and immunohistochemical analyses were performed to compare primary and different generations of PDX tumors. DNA and RNA sequencing were performed to verify the similarity between the genetic profiles of primary and PDX tumors. Total RNA in extracellular vesicles (EVs) released from primary and PDX tumors was also quantified to evaluate gene expression. The total tumor engraftment rate was 50%. Histologically, no major differences were observed between the original and PDX tumors. Most of the gene mutations and expression patterns related to carcinogenesis and infiltration were similar between the primary tumor and xenograft. Most genes associated with carcinogenesis and infiltration showed similar expression levels in the primary tumor and xenograft EVs. Therefore, compared with primary tumors, PDX models could be potentially more useful for translational research.

Abstract 5187: Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment

Abstract NK cells are highly potent, fast-acting cytolytic cells capable of eradicating cancer with limited risk of adverse effects such as cytokine release syndrome or GVHD. NKX101 is a NK cell therapy product engineered to overexpress a chimeric receptor consisting of NKG2D ectodomain, costimulatory signaling motifs, and a membrane-bound form of IL-15. NKX101 is currently under clinical evaluation for treatment of relapsed/refractory AML and MDS. To better understand patterns of response to NKX101, we describe the development of (i) a single-cell (sc) RNAseq approach to assess gene expression pattern changes in NKX101 and patient cells, (ii) a multiplex IHC panel to monitor NKG2D-ligand expression by cancer cells, and (iii) an ELISA method to detect NKG2D-ligand shedding in serum. To verify that our scRNAseq approach can distinguish the NKX101 product cells from patient cells, healthy human PBMCs were pre-mixed with NKX101 cells at set ratios. 10x Genomics hybrid capture baits were prepared to enrich for exogene reads, first via single-cell 5’ gene expression assays and then further processed for target enrichment using a human immunology gene set panel. Multiplex IHC was carried out using the Vectra Polaris Phenoptics platform. FFPE biopsy samples were stained with an 8-marker multiplex panel. Digital image analysis was conducted using the inForm analysis software. For the detection of shed NKG2D-ligands, a sandwich ELISA was employed using recombinant human NKG2D to capture the ligands, followed by detection using ligand-specific antibodies and HRP-conjugated secondary antibodies. We can successfully separate NKX101 product cells from the host PBMC background using our scRNAseq data. NKX101 cell proportions in the transcriptome datasets correlated with the expected titration ratios against host PBMC, with samples titrated at 25% NKX101+ cells yielding approximately 25% NKX101 exogene expressing cells by scRNAseq. Our internally developed multiplex IHC methods can quantify NKG2D-ligand expression levels in AML and solid tumors. We show examples of NKG2D-ligand expression on blast cells from AML solid bone marrow biopsies. In solid tumors, we demonstrate that NKG2D-ligands are upregulated on PanCK+ cancer cells compared to PanCK- cells or healthy normal tissues. Finally, we found certain commercial kits for the detection of soluble NKG2D ligands to potentially under-report the NKG2D-ligand concentrations due to their propensity for aggregation. Using recombinant NKG2D as a capture reagent, we have developed sandwich ELISA method to detect NKG2D-ligands shed into the culture supernatant or serum. In summary, we describe here the development of IHC and ELISA methods to determine NKG2D-ligand expression and shedding as well as a scRNAseq assay to identify NKX101+ product cells and evaluate the anti-tumor immune response during CAR-NK treatment. Citation Format: Kyle Hansen, Sombeet Sahu, Bao Duong, Cynthia Cho, Qi Zhang, James Trager, Joanne Tan. Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5187.

Abstract 5724: Loss of SWI/SNF chromatin remodeling alters NRF2 signaling in human cancers

Abstract Next-generation sequencing studies of lung cancer has shown a significant number of mutations in two previously unrecognized regulatory pathways. Activating mutations, the NF-E2-related factor 2 (NFE2L2, NRF2) transcription factor induces constitutive expression of cytoprotective and redox maintenance genes. Inactivating mutations in members of the SWI/SNF chromatin-remodeling complex, especially SMARCA4 (BRG1) and ARID1A, lead to changes in nucleosome positioning and gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression and protein levels of the NRF2-target genes HMOX1 and GSTM4. RNA sequencing of BRG1-proficient and -deficient cell lines confirmed changes in KEAP1-NRF2 target gene expression as well as identified novel targets of the SWI/SNF complex. Importantly, low BRG1 expression levels in primary human NSCLC tumors correlated with increased NRF2-target gene expression. In addition to BRG1 levels, we examined loss of ARID1A across primary human tumors and found ARID1A deficiency was associated with altered NRF2 signaling. The tumors with the highest frequency of altered NRF2 signaling were lung squamous (LUSC) and head and neck squamous (HNSCC), cancers strongly associated with environmental toxicants. Interestingly ARID1A loss depressed NRF2 signaling in LUSC while amplifying it in HNSCC, an observation we are exploring mechanistically in cell line knock out models. Our data demonstrate that loss of SWI/SNF in lung cancer results in activation of the NRF2/KEAP1 pathway and increased expression of a subset of its downstream targets. These data provide an additional molecular explanation for why patients harboring SWI/SNF mutations show a poor prognosis. Our results also potentially link mutations in SWI/SNF complex components, that occur in >20% of human cancers, with exposure to environmental toxicants. A better understanding of these mechanism may yield novel insights into the design of targeted treatment modalities for human lung cancers. Citation Format: Vinh Nguyen, Travis Shranck, Samera Hamad, Bernard Weissman. Loss of SWI/SNF chromatin remodeling alters NRF2 signaling in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5724.

Abstract 1414: Assessment of the Cancer Testis antigen AKAP4, via tumor expression analysis and IHC, as a potential vaccine and immunotherapy target for colorectal cancer

Abstract Introduction: Cancer-Testis antigens (CTA’s) are normally expressed in germline tissue, however, aberrant expression occurs in some cancers. Restricted CTA expression in normal tissues make CTA’s potential vaccine targets. CTA’s have been associated with tumor cell proliferation and invasion. The CTA A-kinase anchor proteins 4 (AKAP 4) plays a role in cancer development and progression. The expression pattern of AKAP4 gene in CRC is not well characterized. This study’s aim was to assess AKAP4 expression in CRC and to assess this CTA’s potential as a vaccine target. Methods: CRC patients (pts) undergoing surgery who donated tissue to an IRB approved tissue/data bank comprise the study group. Demographic and pathologic data were assessed. Tissues were OCT embedded and stored at -800C. Total purified RNA was isolated from tissue samples and cDNA synthesized. AKAP4 expression was analyzed by quantitative PCR (QPCR) using EXPRESS qPCR universal Supermix and Taqman assay. Comparative quantitative analysis was performed based on the delta-delta Ct method with GAPDH as internal control. Tumor and testis AKAP4 expression levels were determined; tumors with levels 0.1% or more than the testis was considered positive. Immunohistochemistry (IHC) was performed on a subset of tumor and normal tissue. The impact of tumor location and cancer stage on AKAP4 expression was determined and assessed (Wilcoxon signed rank test). Results: 70 paired CRC and normal tissue specimens (35 M/36 F, age 67.8±14) were studied (86% colon,14% rectal; cancer stage: 1, 24%; 2,37%; 3, 31.%;stg 4, 7%). The percent of pts with a relative Malignant to Normal tissue AKAP4 expression ratio (MN) over 1 was 17%; the percent with both MN ratio over 1 and expression levels above 0.1% of testis levels was 16%. IHC demonstrated AKAP4 in a subset of CRC tumor samples. No significant difference was noted between colon vs rectal, or cancer stage groups. No expression was found in 21 normal samples. Discussion: In a tumor subset the relative expression of AKAP4 was above that of normal colon and more than 0.1% of testis expression. IHC study demonstrated the antigen in CRC tumors. A larger study is needed determine if AKAP4 expression correlates with T, N, or final tumor stage. AKAP4 holds some promise as a vaccine target. Citation Format: Chandana S. K. Herath Mudiyanselage, Neil Mitra, Otavia L. Caballero, Dasuni N. Gamage, Xiaohong Yan, Vesna Cekic, Joseph Martz, Richard L. Whelan. Assessment of the Cancer Testis antigen AKAP4, via tumor expression analysis and IHC, as a potential vaccine and immunotherapy target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1414.

Methylation Status of Gene Bodies of Selected microRNA Genes Associated with Neoplastic Transformation in Equine Sarcoids

Horses are of great importance in recreation, livestock production, as working animals in poorly developed countries, and for equine-assisted therapy. Equine sarcoids belong to the most commonly diagnosed tumors in this species. They may cause discomfort, pain, and can lead to the permanent impairment of motor function. The molecular bases of their formation are still under investigation. Our previous studies revealed altered microRNA (miRNA) expression and DNA methylation levels in sarcoid tumors. Abnormal patterns of methylation may be responsible for changes in gene expression levels, including microRNAs. Recently, the DNA methylation of gene bodies has also been shown to have an impact on gene expression. Thus, the aim of the study was to investigate the methylation pattern of gene bodies of chosen miRNAs identified in sarcoid tissue (miR-101, miR-10b, miR-200a, and miR-338-3p), which have also been established to play roles in neoplastic transformation. To this end, we applied qRT-PCR, Bisulfite Sequencing PCR (BSP), and Mquant methods. As a result, we identified the statistically significant downregulation of pri-mir-101-1, pri-mir-10b, and pri-mir-200a in the sarcoid samples in comparison to the control. The DNA methylation analysis revealed their hypermethylation. This suggests that DNA methylation may be one mechanism responsible for the downregulation of theses miRNAs. However, the identified differences in the methylation levels are not very high, which implies that other mechanisms may also underlie the downregulation of the expression of these miRNAs in equine sarcoids. For the first time, the results obtained shed light on microRNA expression regulation by gene body methylation in equine sarcoids and provide bases for further deeper studies on other mechanisms influencing the miRNA repertoire.

Identification of response stratification factors from pooled efficacy analyses of afamitresgene autoleucel (“Afami-cel” [Formerly ADP-A2M4]) in metastatic synovial sarcoma and myxoid/round cell liposarcoma phase 1 and phase 2 trials.

11562 Background: Afami-cel is an autologous, HLA-A*02-restricted, specific peptide enhanced affinity receptor, T-cell therapy engineered to target MAGE-A4+ solid tumors. The pivotal, 2-cohort, single-arm, Phase 2, SPEARHEAD-1 trial (NCT04044768) with afami-cel met its primary endpoint based on Cohort 1 data. As of September 1, 2021, in 47 patients (pts) with metastatic synovial sarcoma (SyS) or myxoid/round cell liposarcoma (MRCLS), the overall response rate (ORR) per independent review was 34% with encouraging durability (Van Tine, et al. Paper 30: CTOS 2021; Virtual). To identify potential stratification factors for response and assess whether response is a proxy for progression-free survival (PFS), we present pooled analyses using data from the prior Phase 1 trial (NCT03132922) and Cohort 1 of the SPEARHEAD-1 trial. Methods: Eligible pts (16–75 years) were HLA-A*02+ with MAGE-A4+ tumors. Pts received afami-cel after lymphodepleting chemotherapy. The pooled analyses evaluated ORR per RECIST v1.1 by investigator review, stratified by 7 factors, and safety. Results: In the pooled data, 69 pts received afami-cel (2.12–9.99×109 transduced T-cells) and were evaluable for response (Phase 1, n = 18; Phase 2, n = 51); all expressed one eligible HLA-A*02 allele. Median (range) for: age was 42 years (19–76), number of prior lines of therapy was 2 (1–12), and tumor MAGE-A4 H-score was 230 (60–300). Median (range) H-score was higher in SyS (256 [60–300]) than in MRCLS (180 [112–230]). The pooled investigator-assessed ORR was 36.2% (40.7% in SyS; 10.0% in MRCLS). Responses occurred across a wide MAGE-A4 H-score range (134–300). Median (range) duration of response was 52 weeks (8.29–75.14). Response rate was higher in the 59 pts with SyS: with ≤2 vs ≥3 prior lines of therapy (55.2% vs 26.7%), baseline target lesion sum of longest diameters <10cm vs ≥10cm (53.1% vs 25.9%), MAGE-A4 H-score ≥200 vs <200 (46.3% vs 27.8%), without vs with bridging therapy (48.6% vs 29.2%), who were female vs male (46.4% vs 35.5%), aged ≥40 vs <40 years (45.7% vs 33.3%), and from North America vs Europe (42.6% vs 33.3%). In responders vs non-responders with SyS, respectively, median PFS was 58.3 vs 11. 0 weeks (log-rank p-value <0.0001); the probability of being progression-free at 24 weeks was 0.8 vs 0.2. The pooled benefit:risk profile of afami-cel was similar to that in the SPEARHEAD-1 trial (Van Tine, et al. Paper 30: CTOS 2021; Virtual.). Conclusions: We show that baseline tumor burden, prior systemic treatment history, and MAGE-A4 tumor expression levels are potential factors associated with response to afami-cel, although their true predictive value for response status awaits confirmation. Our findings will inform the ongoing clinical development of afami-cel in sarcoma, especially for prognostic studies with PFS or overall survival endpoints. Clinical trial information: NCT04044768, NCT03132922.

The K-Ras(G12D)-inhibitory peptide KS-58 suppresses growth of murine CT26 colorectal cancer cell-derived tumors

Mutations in the cell proliferation regulator K-Ras are found with a variety of cancer types, so drugs targeting these mutant proteins could hold great clinical potential. Very recently, a drug targeting the K-Ras(G12C) mutant observed in lung cancer gained regulatory approval and several clinical trials are currently underway to examine the efficacy of this agent when combined with other drugs such as a monoclonal antibody inhibitor of programmed cell death 1 receptor (anti-PD-1). Alternatively, there are currently no approved drugs targeting K-Ras(G12D), the most common cancer-associated K-Ras mutant. In 2020, we described the development of the K-Ras(G12D) inhibitory bicyclic peptide KS-58 and presented evidence for anticancer activity against mouse xenografts derived from the human pancreatic cancer cell line PANC-1 stably expressing K-Ras(G12D). Here, we show that KS-58 also possess anticancer activity against mouse tumors derived from the colorectal cancer cell line CT26 stably expressing K-Ras(G12D). Further, KS-58 treatment reduced phosphorylation of ERK, a major downstream signaling factor in the Ras pathway, confirming that KS-58 inhibits K-Ras(G12D) function. Unexpectedly; however, KS-58 did not show additive or synergistic anticancer activity with mouse anti-PD-1. Morphological analysis and immunostaining demonstrated no obvious differences in CD8+ cells infiltration or PD-L1 expression levels in CT26-derived tumors exposed to monotherapy or combination treatment. Nonetheless, KS-58 demonstrated reasonable stability in blood (t1/2 ≈ 30 min) and no obvious systemic adverse effects, suggesting clinical potential as a lead molecule against colorectal cancer.

Identification of miRNA biomarkers for stomach adenocarcinoma

BackgroundStomach adenocarcinoma (STAD) is a common malignant tumor in the world and its prognosis is poor, miRNA plays a role mainly by influencing the expression of mRNAs, and participates in the occurrence and development of tumors. However, reliable miRNA prognostic models for stomach adenocarcinoma remain to be identified.ResultsUsing the data from the Cancer Genome Atlas (TCGA), a prognostic model of stomach adenocarcinoma was established including tumor stage and expression levels of 4 miRNAs (hsa-miR-379-3p, hsa-miR-2681-3p, hsa-miR-6499-5p and hsa-miR-6807-3p). A total of 50 ultimate target genes of these miRNAs were obtained through prediction. Enrichment analysis revealed that target genes were mainly concentrated in neural function and TGF-β and FoxO signaling pathways. Survival analysis showed that three model miRNAs (hsa-miR-379-3p, hsa-miR-2681-3p and hsa-miR-6807-3p) and five final target genes (DLC1, LRFN5, NOVA1, POU3F2 and PRICKLE2) were associated with the patient's overall survival outcome.ConclusionsWe used bioinformatics methods to screen new prognostic miRNA markers from TCGA and established a prognostic model of STAD, so as to provide a basis for the diagnosis, prognosis, and treatment of STAD in the future.

HSF1 Signaling Inhibits Apoptosis in Hepatoblastoma

IntroductionHepatoblastoma (HB) is the most common pediatric liver malignancy. While relatively rare, its incidence is increasing. Currently surgical resection with or without chemotherapy is the main treatment for the tumor though in severe cases children require liver transplantation. Development of precision therapy for the tumor has been hampered by gaps in fundamental knowledge about how the tumor develops and grows. Heat shock factor 1 (HSF1) is the master regulator of the heat shock response, which classically mediates cellular stress from heat but it likely has a cancer specific. Recent work has shown that HSF1 controls a suite of genes in cancer related to apoptosis, cell cycle signaling, DNA repair, energy metabolism and extracellular matrix formation. Previously, we conducted a meta‐analysis of six human HB transcriptomic data sets, which found expression levels of HSF1 was higher in tumors versus normal liver. Intriguingly, we found higher levels of HSF1 expression in tumors correlated with mortality. We wanted to investigate how inhibiting HSF1 impacted tumor growth in our lab’s mouse model of hepatoblastoma based on transfection with constitutively active forms of β‐catenin and yes‐associated protein 1 (Yap1). This model induces tumors that genetically and histologically resemble human hepatoblastoma. The objective of this study was to investigate the specific role of HSF1 signaling in the development of hepatoblastoma. Our hypothesis is inhibiting HSF1 in our mouse model of hepatoblastoma leads to increased apoptosis in tumor cells and thus decreased tumor size and number.MethodsWe inhibited HSF1 signaling in mice by co‐transfecting a dominant negative HSF1 (dnHSF1) plasmid along with β‐catenin and Yap1 plasmids. The dnHSF1 forms a truncated form of the protein that cannot initiate transcription of target genes. Mice injected with just β‐catenin and Yap1 plasmids served as controls. Eight weeks after transfection, the mice were harvested and the livers were preserved for analysis. Liver and body weights of each mouse was measured to calculate liver‐to‐body weight ratio, an index of tumor burden. At least three mice per condition was assessed.ResultsWe found transfection with the dnHSF1 lead to decreased liver‐to‐body weight ratio compared to control mice transfected with just the β‐catenin and Yap1 plasmids (p‐value<0.05)(Figure 1). What few tumor foci remained featured larger, more differentiated cells. Indeed, these foci showed positive staining via immunohistochemistry (IHC) for both activated mTORC1 and glutamine synthetase, markers seen early in tumor development. Next, we assessed apoptosis by conducting IHC for cleaved caspase 3 and by performing a TUNEL assay on liver sections from both dnHSF1/β‐catenin/Yap1 mice and from control β‐catenin/Yap1 animals. Staining for both markers was increased nearly three times in dnHSF1 treated animals versus control(p‐value<0.05)(Figure 2).ConclusionsWe conclude inhibiting HSF1 signaling in burgeoning hepatoblastoma cells leads to smaller, more differentiated tumors via increased apoptosis. More work is needed to determine if HSF1 is a viable pharmacologic target for hepatoblastoma.

Dietary patterns in association with the expression of pro-metastatic genes in primary breast cancer.

Metastasis is a major leading cause of mortality in female breast cancer (BrCa). Cellular motility is a pathological process of metastasis remarked by the overexpression of cortactin (CTTN), Ras homolog family member-A (RhoA), and Rho-associated kinase (ROCK) genes.Their balanceisresponsible forupholding the integrity of healthy epithelial cell junctions. This study aimed to explore the associations between a posteriori dietary patterns and theexpression levels of pro-metastatic genes in primary BrCa. In this consecutive case series, 215 eligible women, newly diagnosed with histologically confirmednon-metastatic BrCa (stage I-IIIA), were recruited from Hospitals in Tabriz, NorthwesternIran (2015-2017). The tumoral expression levels of genes were quantified using real-time reverse transcription-polymerase chain reaction. Dietary data assessment was carried out using a validated food frequency questionnaire. Three dietary patterns were identified using principal component analysis (KMO = 0.699). Adherence to the "vegan" pattern (vegetables, fruits, legumes, nuts, seeds, and whole grains) was inversely associated with the expression levels of RhoA (ORAdj.T3vs.T1 = 0.24, 95%CI 0.07-0.79) and ROCK (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.87). In addition, the highest adherence to the "prudent" pattern (spices, seafood, dairy, and vegetable oils) decreased the odds of overexpressions at RhoA (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.84) and ROCK genes (ORAdj.T3vs.T1 = 0.29, 95%CI 0.09-0.95). The highest adherence to "Western" pattern (meat, processed meat, hydrogenated fat, fast food, refined cereals, sweets, and soft drinks) was a risk factor associated with the overexpression of RhoA (ORAdj.T3vs.T1 = 3.15, 95%CI 1.12-8.85). Adherence to healthy dietary patterns was significantly associated with the downregulation of pro-metastatic genes. Findings provided new implications to advance the nutrigenomic knowledge to prevent the odds ofover-regulations in pro-metastatic genes of theprimary BrCa.

Fanconi Anemia Pathway in Colorectal Cancer: A Novel Opportunity for Diagnosis, Prognosis and Therapy.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and has the second highest mortality rate globally. Thanks to the advent of next-generation sequencing technologies, several novel candidate genes have been proposed for CRC susceptibility. Germline biallelic mutations in one or more of the 22 currently recognized Fanconi anemia (FA) genes have been associated with Fanconi anemia disease, while germline monoallelic mutations, somatic mutations, or the promoter hypermethylation of some FANC genes increases the risk of cancer development, including CRC. The FA pathway is a substantial part of the DNA damage response system that participates in the repair of DNA inter-strand crosslinks through homologous recombination (HR) and protects genome stability via replication fork stabilization, respectively. Recent studies revealed associations between FA gene/protein tumor expression levels (i.e., FANC genes) and CRC progression and drug resistance. Moreover, the FA pathway represents a potential target in the CRC treatment. In fact, FANC gene characteristics may contribute to chemosensitize tumor cells to DNA crosslinking agents such as oxaliplatin and cisplatin besides exploiting the synthetic lethal approach for selective targeting of tumor cells. Hence, this review summarizes the current knowledge on the function of the FA pathway in DNA repair and genomic integrity with a focus on the FANC genes as potential predisposition factors to CRC. We then introduce recent literature that highlights the importance of FANC genes in CRC as promising prognostic and predictive biomarkers for disease management and treatment. Finally, we represent a brief overview of the current knowledge around the FANC genes as synthetic lethal therapeutic targets for precision cancer medicine.

Role of Furin in Colon Cancer Stem Cells Malignant Phenotype and Expression of LGR5 and NANOG in KRAS and BRAF-Mutated Colon Tumors.

Simple SummaryColorectal cancer (CRC) is one of the most common malignancies in the digestive system. We have previously shown that the proprotein convertase Furin is involved in calcium regulation in cancer cells. In this study, we revealed that the malignant phenotype of colon cancer stem cells is repressed by Furin inhibition that is associated with reduced expression of LGR5 and Nanog and dysregulated expression of several calcium regulators involved in colon cancer. Our data support the idea that targeting Furin in colorectal cancer stem cells may constitute a potential therapeutic approach.Proprotein convertases or PCs are known to regulate the malignant phenotype of colon cancer cells by different mechanisms, but their effects on cancer stem cells (CSCs) have been less widely investigated. Here, we report that PCs expression is altered in colon CSCs, and the inhibition of their activity reduced colon CSCs growth, survival, and invasion in three-dimensional spheroid cultures. In vivo, repression of PCs activity by the general PC inhibitors α1-PDX, Spn4A, or decanoyl-RVKR-chloromethylketone (CMK) significantly reduced tumor expression levels of the stem cell markers LGR5 and NANOG that are associated with reduced tumor xenografts. Further analysis revealed that reduced tumor growth mediated by specific silencing of the convertase Furin in KRAS or BRAF mutated-induced colon tumors was associated with reduced expression of LGR5 and NANOG compared to wild-type KRAS and BRAF tumors. Analysis of various calcium regulator molecules revealed that while the calcium-transporting ATPase 4 (ATP2B4) is downregulated in all the Furin-silenced colon cancer cells, the Ca2+-mobilizing P2Y receptors, was specifically repressed in BRAF mutated cells and ORAI1 and CACNA1H in KRAS mutated cells. Taken together, our findings indicate that PCs play an important role in the malignant phenotype of colon CSCs and stem cell markers’ expression and highlight PCs repression, particularly of Furin, to target colon tumors with KRAS or BRAF mutation.

Exploring Immune-Related Prognostic Signatures in the Tumor Microenvironment of Colon Cancer.

Background: Colon cancer is a common malignant tumor with poor prognosis. The aim of this study is to explore the immune-related prognostic signatures and the tumor immune microenvironment of colon cancer. Methods: The mRNA expression data of TCGA-COAD from the UCSC Xena platform and the list of immune-related genes (IRGs) from the ImmPort database were used to identify immune-related differentially expressed genes (DEGs). Then, we constructed an immune-related risk score prognostic model and validated its predictive performance in the test dataset, the whole dataset, and two independent GEO datasets. In addition, we explored the differences in tumor-infiltrating immune cell types, tumor mutation burden (TMB), microsatellite status, and expression levels of immune checkpoints and their ligands between the high-risk and low-risk score groups. Moreover, the potential value of the identified immune-related signature with respect to immunotherapy was investigated based on an immunotherapeutic cohort (Imvigor210) treated with an anti-PD-L1 agent. Results: Seven immune-related DEGs were identified as prognostic signatures. The areas under the curves (AUCs) of the constructed risk score model for overall survival (OS) were calculated (training dataset: 0.780 at 3 years, 0.801 at 4 years, and 0.766 at 5 years; test dataset: 0.642 at 3 years, 0.647 at 4 years, and 0.629 at 5 years; and the whole dataset: 0.642 at 3 years, 0.647 at 4 years, and 0.629 at 5 years). In the high-risk score group of the whole dataset, patients had worse OS, higher TMN stages, advanced pathological stages, and a higher TP53 mutation rate (p < 0.05). In addition, a high level of resting NK cells or M0 macrophages, and high TMB were significantly related to poor OS (p < 0.05). Also, we observed that high-risk score patients had a high expression level of PD-L1, PD-1, and CTLA-4 (p < 0.05). The patients with high-risk scores demonstrated worse prognosis than those with low-risk scores in multiple datasets (GSE39582: p = 0.0023; GSE17536: p = 0.0008; immunotherapeutic cohort without platinum treatment: p = 0.0014; immunotherapeutic cohort with platinum treatment: p = 0.0027). Conclusion: We developed a robust immune-related prognostic signature that performed great in multiple cohorts and explored the characteristics of the tumor immune microenvironment of colon cancer patients, which may give suggestions for the prognosis and immunotherapy in the future.

Abstract P1-07-02: Primary results of ONAWA (SOLTI-1802) trial: A window of opportunity trial of onapristone in postmenopausal women with progesterone receptor-positive/HER2-negative early breast cancer (EBC)

Abstract Background PgR expression is a biomarker of ER functionality, cellular progression to malignancy, and response to endocrine therapy (ET) in HR+ BC. Onapristone (ONA), a type 1 antiprogestin, was shown to have a single agent anti-tumor activity in patients with metastatic breast cancer (Robertson et al., 1999; Jonat et al., 2002). However, this once daily immediate-release formulation was associated with liver function test abnormalities in one-third of patients. A new, extended-release formulation (ONA XR) was developed and was evaluated in a BID schedule that reduced peak serum concentrations while sustaining the minimum plasma concentrations previously associated with the higher dose. Safety results of two phase I-II studies confirmed this hypothesis (Cottu et al., 2018; Jayaram et al., 2017; Lewis et al, 2020). Considering BC heterogeneity and that PgR analysis by standard immunohistochemistry (IHC) does not perfectly correlate with PgR target gene expression, the identification of biomarkers allowing the selection of patients with PgR-driven tumors that may benefit from antiprogestins treatment is currently an unmet need. Patients and Methods ONAWA (NCT04142892) is an open-label, single-arm, multicenter window of opportunity clinical trial of ONA XR (50 mg BID for 21 days) for postmenopausal women with EBC amenable to receive a short course of ET before surgery. Ten patients with ER+/PgR+/HER2- and Ki-67 ≥ 15% BC were enrolled. The primary objective is to evaluate the biological activity of ONA by the rate of Complete Cell Cycle Arrest (CCCR) determined by Ki-67 (≤2.7%). Secondary endpoints include safety and correlating biological activity with IHC of tumor expression (ER, PgR, Ser294-PgR, CD24, CD44, ALDH1, Ki-67), estradiol, and progesterone blood levels, and gene expression profile (NanoString nCounter® Breast 360TM panel). Relative Ki-67 suppression was defined as (1 - Ln(Ki-67 Baseline)/Ln(Ki-67 surgery)) Results Assessment of the treatment effects was possible for the 10 patients who successfully completed the protocol and the 10 paired samples (100%) were analyzed. Main patient characteristics were mean age 68 (range 53-81 years), mean tumor size 20.2 mm (range 15-26 mm), stage I (40%) and grade 2 (100%). No patients achieved a CCCR. Tumor Ki-67 expression decreased in six patients was stable in one patient and increased three patients. The mean percentage suppression of Ki-67 was 19.58%. Overall, no statistically significant change was observed in Ki-67 between paired samples (p=0.234). Baseline IHC PgR (%) expression correlated with Ki-67 decrease (r = -0.635). Mean percentage suppression of Ki-67 for tumors with IHC PgR expression ≥90% (N=4) and &amp;lt;90% (N=6) was -25.23.0% and +2.54%, respectively. Six (60%) patients reported AEs at any grade. Most common grade 1 or 2 AEs were post-procedural pain, dry mouth and GGT increased. Grade 3 reversible GGT and AST increase occurred in 1 patient. Conclusion: ONA XR significantly increases suppression of tumor cell proliferation in PgR-high primary breast cancer. The safety profile was consistent with that previously reported. Additional correlative analysis including gene expression will be presented. Acknowledgments: Funding and drug provided by Context Therapeutics Inc., USA. Citation Format: Meritxell Bellet, Serafin Morales, Ariadna Gasol, Kepa Amillano, Nuria Chic, Xavier González-Farré, Patricia Villagrasa, Laia Paré, Claudette Falato, Paolo Nuciforo, Débora Martínez, Juan M Ferrero-Cafiero, Tomás Pascual, Aleix Prat, Carol Lange, Cristina Saura. Primary results of ONAWA (SOLTI-1802) trial: A window of opportunity trial of onapristone in postmenopausal women with progesterone receptor-positive/HER2-negative early breast cancer (EBC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-07-02.

Downstream of the HOX genes: Explaining conflicting tumour suppressor and oncogenic functions in cancer.

The HOX genes are a highly conserved group of transcription factors that have key roles in early development, but which are also highly expressed in most cancers. Many studies have found strong associative relationships between the expression of individual HOX genes in tumours and clinical parameters including survival. For the majority of HOX genes, high tumour expression levels seem to be associated with a worse outcome for patients, and in some cases, this has been shown to result from the activation of pro‐oncogenic genes and pathways. However, there are also many studies that indicate a tumour suppressor role for some HOX genes, sometimes with conclusions that contradict earlier work. In this review, we have attempted to clarify the role of HOX genes in cancer by focusing on their downstream targets as identified in studies that provide experimental evidence for their activation or repression. On this basis, the majority of HOX genes would appear to have a pro‐oncogenic function, with the notable exception of HOXD10, which acts exclusively as a tumour suppressor. HOX proteins regulate a wide range of target genes involved in metastasis, cell death, proliferation and angiogenesis, and activate key cell signalling pathways. Furthermore, for some functionally related targets, this regulation is achieved by a relatively small subgroup of HOX genes.