Metastatic Gene Research Articles

P10.12.B MOLECULAR MECHANISMS INVOLVED IN THE DEVELOPMENT OF BRAIN METASTASIS FROM THE MATCHED PAIRED LUNG ADENOCARCINOMA AND BREAST CANCER

Abstract BACKGROUND Up to 50% of patients with common cancers will develop brain metastases and incidences of this devastating complication are still rising. In our recent work, we found that the immune response in the brain is significantly stronger when the metastasis develops from lung adenocarcinoma (LUAD) compared to breast cancer (BC). Importantly, we discovered the higher expression of the drug target, VISTA, only in the brain metastasis of the LUAD. The aim of the current study was to investigate the molecular mechanisms driving the formation of brain metastasis from the two different lineages of cancer. In addition, we aimed to examine the expression level of the drugable molecules between primary tumors and their brain metastasis. MATERIAL AND METHODS Primary tumor and their matched brain metastasis samples of 11 LUAD and 11 breast cancer (total = 44 samples) were included in this study. RNA was extracted from the FFPE samples and the targeted gene expression profiles were measured using the PanCancer IO360™ Panel that includes 770 cancer-related genes (NanoString technology). Data were analysed using the nSolver software. RESULTS A first comparison was made between the primary tumors with their brain metastases for LUAD and BC separately. The second comparison was made between the common genes of both lineages. Pathway analysis indicated that there is higher metabolic stress in breast cancer-brain metastasis compared to that in the lung. In addition, we identified 12 common up-regulated genes in brain metastasis, despite the origin of the primary tumor. Importantly, by comparing the primary tumors of BC and LUAD with their brain metastasis, we found that VISTA is predominantly highly expressed in the primary tumors of LUAD and their matched brain metastasis. CONCLUSION Although the general mechanisms of metastasis have been described, our study demonstrates that different cancer lineages utilize various genes/pathways to metastasize to the brain. Moreover, primary LUAD and their brain metastasis express high levels of VISTA highlighting the possibilities for targeted therapy.

Predicting cervical lymph node metastasis in OSCC based on computed tomography imaging genomics.

To investigate the correlation between computed tomography (CT) radiomic characteristics and key genes for cervical lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC). The region of interest was annotated at the edge of the primary tumor on enhanced CT images from 140 patients with OSCC and obtained radiomic features. Ribonucleic acid (RNA) sequencing was performed on pathological sections from 20 patients. the DESeq software package was used to compare differential gene expression between groups. Weighted gene co-expression network analysis was used to construct co-expressed gene modules, and the KEGG and GO databases were used for pathway enrichment analysis of key gene modules. Finally, Pearson correlation coefficients were calculated between key genes of enriched pathways and radiomic features. Four hundred and eighty radiomic features were extracted from enhanced CT images of 140 patients; seven of these correlated significantly with cervical LNM in OSCC (p < 0.01). A total of 3527 differentially expressed RNAs were screened from RNA sequencing data of 20 cases. original_glrlm_RunVariance showed significant positive correlation with most long noncoding RNAs. OSCC cervical LNM is related to the salivary hair bump signaling pathway and biological process. Original_glrlm_RunVariance correlated with LNM and most differentially expressed long noncoding RNAs.

Synthesis Folate-linked Chitosan-coated Quetiapine/BSA Nano-Carriers as the Efficient Targeted Anti-Cancer Drug Delivery System.

Quetiapine (QTP) has been known to suppress cancer progression in patients suffering from mental disorders. This study aimed to produce the folate-linked chitosan-coated quetiapine/BSA nano-carriers (FCQB-NCs) and evaluate their antioxidant, apoptotic, and anti-metastatic potentials on prostate, pancreas, colon, and breast cancer cell lines. The FCQB-NCs were designed, produced, and characterized using DLS, FESEM, FTIR, and Zeta potential techniques. The nano-carriers antioxidant activity was studied by applying ABTS, DPPH, and FRAP assays. The FCQB-NCs' cytotoxicity and apoptotic/metastatic properties were evaluated utilizing MTT assay and qPCR-based analysis for measuring the apoptotic (Nf-KB)/metastatic (MMP2, MMP9, and MMP13) gene expression, respectively. The AO/PI fluorescent cell staining, DAPI staining, and scratch assay methods were conducted to verify the apoptotic and anti-metastatic activities of FCQB-NCs. The 51-nm FCQB-NCs (PDI = 0.26) exhibited antioxidant activity and selectively decreased the MDA-MB-231 cancer cells' viability by inducing Nf-KB overexpression, which caused the apoptosis pathway activation. Moreover, the FCQB-NCs suppressed the MDA-MB-231 cells' metastatic activity by down-regulating the MMP2, MMP9, and MMP13 gene expression, verified by detecting the decreased migration rate. The FCQB-NCs selectively induced apoptosis and suppressed metastasis in the human breast cancer cell line, which can be attributed to the stepwise release of QTP in two primary (extra-cellular release) and secondary (intra-cellular release) phases. The efficient selective cytotoxic impact of FCQB-NCs can be due to the novel stepwise release mechanism of the FCQB-NCs based on the two-phase entrapment of QTP by BSA and chitosan molecules. Therefore, FCQB-NCs have the potential to be used as an efficient selective anti-breast cancer.

Augmented antitumor effects of erlotinib and cabozantinib on A549 non-small cell lung cancer: In vitro and in vivo studies

Non-small cell lung carcinoma is a challenging disease worldwide. This study aims to determine whether combining erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, with cabozantinib, a mesenchymal-epithelial transition factor (c-Met) inhibitor, would have an augmented therapeutic benefit on A549 cells. The combination of erlotinib and cabozantinib (5 µM) inhibited A549 cell viability compared to each monotherapy at ≥ 10 µM as confirmed by the MTT assay. Combination therapy also has a more potent inhibition of cellular migration than monotherapy using the wound-healing assay. Furthermore, mRNA expression analyses for assessing apoptosis, metastasis, and cell cycle-related genes, the results showed that combination therapy significantly inhibits levels of BCL-2, MMP-9, VEGF, and TGF-β while inducing p53, p21, and BAX expression. In terms of oncogenic markers, western blotting analysis showed a significant reduction of BCl-2 expression and elevation in caspase3, p53, and p21 proteins as indicators of cell death via apoptosis. The antitumor in vivo effect of the combination therapy showed significant tumor inhibition compared to monotherapy. In conclusion, combination therapy could be a potential promising strategy to treat non-small cell lung carcinoma.

NEK6 Promotes the Progression of Osteosarcoma Through Activating STAT3 Signaling Pathway by Down-Regulation of miR-26a-5p.

Osteosarcoma is a malignant tumor originating from the skeletal system. There is no effective treatment other than surgery and chemotherapy, which seriously endangers the health of children and adolescents. NEK6 is a novel discovered Serine/Threonine protein kinase that can regulate cell cycle and activate several oncogenic pathways. NEK6 expression in pan-cancer including sarcoma was evaluated using analysis tools of TIMER, UALCNA and GEPIA with TCGA database, and its association with overall survival in patients with sarcoma was also analyzed. TargetScan, tarbase, microT-CDS and Starbase online software were used to predict NEK6-targeted miRNAs, including miR-26a-5p. Tumor tissues from patients with osteosarcoma were collected for NEK6 and miRNA detection using RT-qPCR. NEK6 down-regulated by siRNAs or miR-26a-5p in osteosarcoma cells was detected by RT-qPCR, Western blot and Immunofluorescence staining assays. Effects of NEK6 knockdown on proliferation, migration, invasion and apoptosis of osteosarcoma cells were detected by CCK-8, wound healing, transwell and flow cytometry, respectively. The expressions of STAT3, metastasis and apoptosis-related genes were detected by Western blot. High expression of NEK6 and low expression of miR-26a-5p were lowly expressed in osteosarcoma and they were negative correlation. NEK6 has been confirmed as a direct target for miR-26a-5p. In addition, NEK6 down-regulated by siRNAs or miR-26a-5p led to inhibition of cell proliferation, migration and invasion while promoting cell apoptosis. The levels of phosphorylated STAT3 and metastasis genes (MMP-2, MMP-9) were inhibited, while apoptotic gene Bax was promoted and Bcl2 was inhibited by miR-26a-5p upregulation. NEK6 can promote osteosarcoma progression via activating STAT3 signaling pathway, which is inhibited by miR-26a-5p, suggesting that NEK6 is a potential oncogene and miR-26a-5p is a suppressor of osteosarcoma. The strategy of inhibiting of NEK6 by miR-26a-5p may be an effective approach for osteosarcoma therapy.

GSK-3 Inhibitor Elraglusib Enhances Tumor-Infiltrating Immune Cell Activation in Tumor Biopsies and Synergizes with Anti-PD-L1 in a Murine Model of Colorectal Cancer.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC.

The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer.

Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.

Comparison of genetic variation between primary colorectal cancer and metastatic peritoneal cancer.

Among cancer metastases by primary colorectal cancer (CRC), peritoneal metastasis is the second most common metastatic lesion after liver metastasis. In treating metastatic CRC, it is very important to differentiate targeted-therapy and chemotherapy according to the characteristics of each lesion because the genetic variation of the primary and metastatic lesions are different. However, there are few studies of genetic characteristics on peritoneal metastasis caused by primary CRC, so molecular-level studies are continuously required. We propose an appropriate peritoneal metastasis treatment policy by identifying the genetic characteristics between primary CRC and synchronous peritoneal metastatic lesions. Primary CRC and synchronous peritoneal metastasis samples were analyzed in pairs from six patients using Comprehensive Cancer Panel (409 cancer-related genes, Thermo Fisher Scientific, USA) and next-generation sequencing (NGS). The mutations were commonly found on the KMT2C and THBS1 genes in both primary CRC and peritoneal metastasis. The PDE4DIP gene was mutated in all cases except for on a sample of peritoneal metastasis. As a result of analysis using the mutation database, we confirmed that the gene mutations of primary CRC and the peritoneal metastasis derived from it showed the same tendency, although we did not accompany the gene expression level or epigenetic study. It is thought that the treatment policy through molecular genetic testing of primary CRC can also be applied to peritoneal metastasis treatment. Our study is expected to be the basis for further peritoneal metastasis research.

Combining next-generation hormonal therapy with PARP inhibition in metastatic castration-resistant prostate cancer

In a paradigm of precision oncology, elegant preclinical work showed that inhibition of poly(ADP-ribose) polymerase (PARP) proteins can exploit a deficiency of cancer cells to repair DNA double-strand breaks through homologous recombination repair (HRR), 1 Lord CJ Ashworth A PARP inhibitors: synthetic lethality in the clinic. Science. 2017; 355: 1152-1158 Crossref PubMed Scopus (1411) Google Scholar and the resulting synthetic lethality was first shown to be clinically effective in ovarian cancer carrying a germline deleterious mutation in BRCA1 or BRCA2, key HRR genes. 2 Banerjee S Moore KN Colombo N et al. Maintenance olaparib for patients with newly diagnosed advanced ovarian cancer and a BRCA mutation (SOLO1/GOG 3004): 5-year follow-up of a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2021; 22: 1721-1731 Summary Full Text Full Text PDF PubMed Scopus (92) Google Scholar For prostate cancer, the discovery of alterations in BRCA2 and other HRR-related genes in up to 20% of advanced disease 3 Robinson D Van Allen EM Wu YM et al. Integrative clinical genomics of advanced prostate cancer. Cell. 2015; 161: 1215-1228 Summary Full Text Full Text PDF PubMed Scopus (2077) Google Scholar led to the development of several biomarker-driven trials of PARP inhibitors in patients with metastatic castration-resistant prostate cancer (mCRPC) 4 Abida W Patnaik A Campbell D et al. Rucaparib in men with metastatic castration-resistant prostate cancer harboring a BRCA1 or BRCA2 gene alteration. J Clin Oncol. 2020; 38: 3763-3772 Crossref PubMed Scopus (309) Google Scholar , 5 de Bono JS Mehra N Scagliotti GV et al. Talazoparib monotherapy in metastatic castration-resistant prostate cancer with DNA repair alterations (TALAPRO-1): an open-label, phase 2 trial. Lancet Oncol. 2021; 22: 1250-1264 Summary Full Text Full Text PDF PubMed Scopus (79) Google Scholar , 6 Hussain M Mateo J Fizazi K et al. Survival with olaparib in metastatic castration-resistant prostate cancer. N Engl J Med. 2020; 383: 2345-2357 Crossref PubMed Scopus (289) Google Scholar , 7 Smith MR Scher HI Sandhu S et al. Niraparib in patients with metastatic castration-resistant prostate cancer and DNA repair gene defects (GALAHAD): a multicentre, open-label, phase 2 trial. Lancet Oncol. 2022; 23: 362-373 Summary Full Text Full Text PDF PubMed Scopus (41) Google Scholar and to the regulatory approval of rucaparib and olaparib as monotherapy for the treatment of mCRPC harbouring mutations in BRCA2 and other HRR genes. Talazoparib plus enzalutamide in men with first-line metastatic castration-resistant prostate cancer (TALAPRO-2): a randomised, placebo-controlled, phase 3 trialTalazoparib plus enzalutamide resulted in clinically meaningful and statistically significant improvement in rPFS versus standard of care enzalutamide as first-line treatment for patients with mCRPC. Final overall survival data and additional long-term safety follow-up will further clarify the clinical benefit of the treatment combination in patients with and without tumour HRR gene alterations. Full-Text PDF

Interleukin-22 promotes the proliferation and migration of hepatocellular carcinoma cells via the phosphoinositide 3-kinase (PI3K/AKT) signaling pathway.

Interleukin-22 (IL-22) is a pro-inflammatory cytokine released during the immune response in chronic liver injury. Although IL-22 mediates tissue regeneration, its uncontrolled production may generate a carcinogenic environment resulting in hepatocellular carcinoma (HCC). This study aims to identify the effect of IL-22 on anti-apoptotic and metastatic genes and the molecular pathways responsible for IL-22-mediated hepatic carcinogenesis. Three cancerous liver lines, HepG2, SNU-387, Huh7, and one normal liver line, THLE2, were treated with IL-22. RT-qPCR analysis was conducted to study the role of IL-22 in altering the expression levels of anti-apoptotic genes, MCL-1 and BCL-2, and metastatic genes, MMP-7 and MMP-9. A significant increase in expression levels of these genes was observed after IL-22 treatment. Furthermore, to explore the major pathways involved in IL-22-mediated upregulation of anti-apoptotic and metastatic genes, cells were treated with inhibitors of JAK/STAT and PI3K/AKT pathways along with IL-22. Resultantly, a significant decrease in expression levels of target genes was observed, indicating the involvement of JAK/STAT and PI3K/AKT signaling cascades in IL-22-mediated oncogenesis. Finally, Cell Scratch assay was performed to check the effect of IL-22 and inhibitors of JAK/STAT and PI3K/AKT on the metastatic potential of liver cells. While migration was observed in Huh7 and THLE2 cells treated with IL-22, no migration was observed in cells treated with IL-22 along with JAK/STAT and PI3K/AKT inhibitors. Results indicate that IL-22 encourages metastasis in HCC cells via the JAK/STAT and PI3K/AKT pathways. Results showed that IL-22 upregulates anti-apoptotic and metastatic genes in HCC through JAK/STAT and PI3K/AKT signaling pathways.

Abstract A055: NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells

Abstract The androgen receptor (AR) is a ligand-responsive transcription factor (TF) that binds as a homodimer at FOXA1-pioneered enhancer elements containing a palindromic DNA motif. Prostate cancer (PCa) is highly dependent on the AR enhanceosome complex, and in castration-resistant disease, this dependency is reinforced through alterations in the AR pathway. This centrally involves extensive rewiring of the AR cistrome to gain de novo binding at enhancer sites (aka neo-enhancers) that activate hyperproliferative and metastatic gene programs. However, requisite subunits of the AR neo-enhanceosome and the genes it uniquely activates remain uncharacterized. Here, we CRISPR-engineered an endogenous AR reporter system by fusing the mCherry coding sequence in-frame with the KLK3/PSA gene and conducted a CRISPR knock-out screen to identify druggable chromatin/epigenetic cofactors of AR. Using a sgRNA library covering &amp;gt;200 druggable transcriptional coregulators, ranked alongside BRD4 and TRIM24/28, we identified NSD2 as a novel AR coactivator. NSD2 is an H3K36 di-methyltransferase that activates gene expression through antagonism of the PRC2/EZH2 complex. In PCa cells, genetic inhibition of NSD2 function significantly attenuated the expression of AR targets without affecting the AR protein level, suggesting that NSD2 regulates either AR’s chromatin assembly or trans-activational competence. Cistromic profiling of AR (ChIPseq) in NSD2-null PCa cells showed complete loss of binding at &amp;gt;40,000 genomic sites (~65% of the cistrome), with HOMER motif analyses of NSD2-dependent AR sites showing a higher recurrence of hexameric 5′-AGAACA-3′ AR half-site juxtaposed to the FOXA1, ETS, or HOXB13 motifs. In contrast, NSD2-independent AR sites, a large fraction of which show increased binding in NSD2-null cells, housed the canonical 15-bp palindromic AR motif with two invertedly-oriented half-sites separately recognized by each half of the AR homodimer. Notably, analyses of primary AR cistromes from human tissues revealed AR loading at half-motifs to be 20-30-fold enriched in the tumor-specific AR enhancer circuitry. In phenotypic assays, we found NSD2-deficient PCa cells to lose cancer hallmarks such as colony formation, invasion, and hyper-proliferation. We further developed a potent NSD2 PROTAC, LLC0150, which showed preferential cytotoxicity in AR/FOXA1+ PCa relative to the AR-negative disease, and other cancer and normal cell lines from 22 distinct lineages. In a pan-cancer screen comprising over 100 cell lines, treatment with LLC0150 triggered apoptotic death in AR/FOXA1+ malignancies and NSD2-mutant acute lymphocytic leukemia, and multiple myeloma. Altogether, we identify NSD2 as a novel AR neo-cofactor that assists oncogenic TFs in loading the AR enhanceosome at degenerate, low-affinity AR elements, thereby wiring its cancer-specific gene programs. We develop a novel NSD2 PROTAC that selectively kills AR/FOXA1-driven prostate and NSD2-altered tumors in preclinical models, positioning NSD2 therapeutics for safety and efficacy evaluation in human clinical trials. Citation Format: Abhijit Parolia, Brijesh K. Verma, Sanjana Eyunni, Sweta Aras, Sandra E. Carson, Eleanor Young, Chandan K. Das, James M. George, Reyaz ur Rasool, Prathibha Gajjala, Mohammed Alhusayan, Erick Mitchell-Velasquez, Lanbo Xiao, Jie Luo, Mustapha Jaber, Xuhong Cao, Fengyun Su, Rui Wang, Lianchao Liu, Zhen Wang, Ke Ding, Arul M. Chinnaiyan, Irfan Asangani. NSD2 is a requisite and targetable subunit of the AR/FOXA1 neo-enhanceosome complex in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A055.

Radiomics in colorectal cancer patients.

The main therapeutic options for colorectal cancer are surgical resection and adjuvant chemotherapy in non-metastatic disease. However, the evaluation of the overall adjuvant chemotherapy benefit in patients with a high risk of recurrence is challenging. Radiological images can represent a source of data that can be analyzed by using automated computer-based techniques, working on numerical information coded within Digital Imaging and Communications in Medicine files: This image numerical analysis has been named "radiomics". Radiomics allows the extraction of quantitative features from radiological images, mainly invisible to the naked eye, that can be further analyzed by artificial intelligence algorithms. Radiomics is expanding in oncology to either understand tumor biology or for the development of imaging biomarkers for diagnosis, staging, and prognosis, prediction of treatment response and diseases monitoring and surveillance. Several efforts have been made to develop radiomics signatures for colorectal cancer patient using computed tomography (CT) images with different aims: The preoperative prediction of lymph node metastasis, detecting BRAF and RAS gene mutations. Moreover, the use of delta-radiomics allows the analysis of variations of the radiomics parameters extracted from CT scans performed at different timepoints. Most published studies concerning radiomics and magnetic resonance imaging (MRI) mainly focused on the response of advanced tumors that underwent neoadjuvant therapy. Nodes status is the main determinant of adjuvant chemotherapy. Therefore, several radiomics model based on MRI, especially on T2-weighted images and ADC maps, for the preoperative prediction of nodes metastasis in rectal cancer has been developed. Current studies mostly focused on the applications of radiomics in positron emission tomography/CT for the prediction of survival after curative surgical resection and assessment of response following neoadjuvant chemoradiotherapy. Since colorectal liver metastases develop in about 25% of patients with colorectal carcinoma, the main diagnostic tasks of radiomics should be the detection of synchronous and metachronous lesions. Radiomics could be an additional tool in clinical setting, especially in identifying patients with high-risk disease. Nevertheless, radiomics has numerous shortcomings that make daily use extremely difficult. Further studies are needed to assess performance of radiomics in stratifying patients with high-risk disease.

Identification of key immune genes related to lymphatic metastasis of papillary thyroid cancer via bioinformatics analysis and experimental validation.

The current research aimed to development and validation in signature immune genes for lymphatic metastasis in papillary thyroid cancer (PTC). Weighted correlation network analysis (WGCNA) was performed to identify genes closely correlated with lymphatic metastasis in PTC from TCGA database. Information on immune-related genes (IRGs) was obtained from the ImmPort database. Crossover genes were used with the R package clusterProfiler for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment. Key genes in the protein-protein interaction network of cross-targets were obtained using Cytoscape. Lasso and Random Forest (RF) models were utilized to identify pivotal genes. We constructed a nomogram based on the hub genes. The correlation between hub genes and immune cell infiltration was explored. We collected and assessed clinical samples via immunohistochemistry to detect the expression of hub genes. In total, 122 IRGs were correlated with lymphatic metastases from PTC. There are 10 key IRGs in the protein-protein interaction network. Then, three hub genes including PTGS2, MET, and ICAM1 were established using the LASSO and RF models. The expression of these hub genes was upregulated in samples collected from patients with lymphatic metastases. The average area under the curve of the model reached 0.83 after a 10-fold and 200-time cross-validation, which had a good prediction ability. Immuno-infiltration analysis showed that the three hub genes were significantly positively correlated with resting dendritic cells and were negatively correlated with activated natural cells, monocytes, and eosinophils. Immunohistochemistry results revealed that lymph node metastasis samples had a higher expression of the three hub genes than non-metastasis samples. Via bioinformatics analysis and experimental validation, MET and ICAM1 were found to be upregulated in lymph node metastasis from papillary thyroid carcinoma. Further, the two hub genes were closely correlated with activated natural killer cells, monocytes, resting dendritic cells, and eosinophils. Therefore, these two genes may be novel molecular biomarkers and therapeutic targets in lymph node metastasis from papillary thyroid carcinoma.

O091 Predict factors of hepatocyte growth factor family and bone morphogenetic proteins for breast cancer nodal metastasis

Abstract Introduction Lymph node metastasis is essential for the outcome of patients with breast cancer. Bone morphogenetic proteins (BMP) and hepatocyte growth factor (HGF), key to angiogenesis and metastasis of cancers, were investigated for their roles in the predication of nodal metastasis from breast cancer. Methods Expression of BMP family ligands, BMP receptors, HGF, its receptor MET and regulatory factors of HGF activation including HGF activator (HGFA), HGFA inhibitor 1 (HAI1), HAI2, Matriptase-1 and Matriptase-2 were determined in breast cancers (n=127) using real time quantitative PCR. Transcript levels of these genes were analysed in the breast cancers for their implication in lymphatic involvement using Mann-Whitney test. Predictive potential of those genes for nodal metastasis of these molecules was evaluated using both binary logistic regression and receiver operating characteristic (ROC) with SPSS (Version 27). Results With median as a cut-off value, logistic regression analyses showed that MET, Matriptase-1 and BMP15 were positively associated with nodal metastasis whereas Matriptase-2, BMP3 and HAI1 were inversely correlated with nodal involvement. After integrating these six prospective factors, ROC model returned with a significant value against nodal status (RUC=0.657, p=0.001). With the optimal ROC cut-off value to dichotomise patients, the integrated expression produced a significant prediction of nodal metastasis (p=0.006, HR=2.929). MET and HAI1 was correlated with the lymphangiogenesis marks of LYVE1, podo, prox1. Matriptase-1 was inversely correlated with podo, whilst Matriptase-2 and BMP15 showed a positive correlation with prox1. Conclusion The MET/Matriptase-1/BMP15 and the inversed Matriptase-2/BMP3/HAI1 are connected significantly with lymph node metastasis in breast cancer.

O096 Expression of MLN70 in breast cancer and the connection with skeletal metastasis

Abstract Introduction MLN70 ([metastatic lymph node gene 70 protein], also known as S100A11 [S100 Calcium-Binding Protein A11] or Calgizzarin), a nucleic and cytosolic protein suspected to have a role in regulation of cellular invasion, migration and tubulin polymerisation in cancer cells and has been implicated in cancer progression. The present study explored how MLN70 might have a value in the clinical outcome of the patients. Methods Expression of MLN70 transcripts was quantified in a clinical cohort of breast cancer and was evaluated against the clinical, pathological, hormonal receptor status and clinical outcome of the patients. Results Breast tumour tissues had higher levels of MLN70 than normal tissues and high levels of MLN70 linked to a shorter overall survival of the patients. MLN70 had a significant correlation with three members of the ERBB family including ERBB1 (EGFR), Her2 (ERBB2) and ERBB4. There were no significant different between node positive and node negative tumours (p=0.237). The most striking finding was that MLN70 showed a significant predictive value in identifying patients who subsequently developed skeletal metastasis (RUC=0.793, p&amp;lt;0.0001 by ROC analysis) and high levels of MLN70 was associated with skeletal metastases (p=0.019, Hazard Ratio 10.93 by regression analysis). Furthermore, multivariate analyses using both linear and regression models indicated MLN70 as being an independent factor for bone metastasis (p=0.016) amongst clinical, pathological and hormone receptor status. Conclusion The present study reveals MLN70 as a significant and independent predictive factor for skeletal metastasis of patients with breast cancer and a prospective target in this cancer type.

SYTL2 promotes metastasis of prostate cancer cells by enhancing FSCN1-mediated pseudopodia formation and invasion

BackgroundMetastatic prostate cancer (mPCa) has a poor prognosis with limited treatment options. The high mobility of tumor cells is the key driving characteristic of metastasis. However, the mechanism is complex and far from clarified in PCa. Therefore, it is essential to explore the mechanism of metastasis and discover an intrinsic biomarker for mPCa.MethodsTranscriptome sequencing data and clinicopathologic features of PCa from multifarious public databases were used to identify novel metastatic genes in PCa. The PCa tissue cohort containing 102 formalin-fixed paraffin-embedded (FFPE) samples was used to evaluate the clinicopathologic features of synaptotagmin-like 2 (SYTL2) in PCa. The function of SYTL2 was investigated by migration and invasion assays and a 3D migration model in vitro and a popliteal lymph node metastasis model in vivo. We performed coimmunoprecipitation and protein stability assays to clarify the mechanism of SYTL2.ResultsWe discovered a pseudopodia regulator, SYTL2, which correlated with a higher Gleason score, worse prognosis and higher risk of metastasis. Functional experiments revealed that SYTL2 promoted migration, invasion and lymph node metastasis by increasing pseudopodia formation in vitro and in vivo. Furthermore, SYTL2 induced pseudopodia formation by enhancing the stability of fascin actin-bundling protein 1 (FSCN1) by binding and inhibiting the proteasome degradation pathway. Targeting FSCN1 enabled rescue and reversal of the oncogenic effect of SYTL2.ConclusionsOverall, our study established an FSCN1-dependent mechanism by which SYTL2 regulates the mobility of PCa cells. We also found that the SYTL2-FSCN1-pseudopodia axis may serve as a pharmacological and novel target for treating mPCa.

Somatic mutation profiling, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein expression in HER2-amplified colorectal cancer.

The status of human epidermal growth factor receptor 2 (HER2) for the prognosis in colorectal cancer (CRC) is controversial, and the characteristics of the somatic mutation spectrum, tumor-infiltrating leukocytes, tertiary lymphoid structures and PD-L1 protein are unknown in HER2-amplified colorectal cancer (HACC). In order to explore these characteristics along with their correlation with clinicopathological factors and prognosis in HACC. Samples of 812 CRC patients was collected. After immunohistochemistry (IHC), 59 of 812 were found to be HER2-positive, then 26 of 59 samples were further determined to be HER2 amplification by fluorescence in situ hybridization (FISH). Somatic mutation profiling of HACC was analysed using whole exome sequencing (WES). Multiplex fluorescence immunohistochemistry (mIHC) was used for tumor-infiltrating leukocytes and tertiary lymphoid structures (TLSs), while PD-L1 protein was detected by IHC. Our results indicate that the detection rates of HER2 positivity by IHC and FISH were 7.3% and 3.2% respectively, and HER2 amplification is correlated with distant tumour metastasis. The somatic mutation profiling revealed no differences between HACC and HER2-negative CRC. However, TP 53 strongly correlated with poor prognosis in HACC. Furthermore, tumor-infiltrating T cells and TLSs in the tumor immune microenvironment, as well as PD-L1 expression, were higher in HACC than in HER2-negative controls. However, none of them were associated with the prognosis of HACC. In all, HER2 amplification is correlated with distant metastasis and TP53 gene mutation may be a potential protective mechanism of HACC.

Genes whose expressions in the primary lung squamous cell carcinoma are able to accurately predict the progression of metastasis through lymphatic system, inferred from a bioinformatics analyses

Lymph node metastasis is the most important prognostic factor in patients with lung squamous cell carcinoma. The current findings show that lymph node metastatic tumor cells can arise by programming metastasis in primary tumor cells. Thereby, the genetic alterations responsible for the metastasis could be detected in the primary tumors. This bioinformatic study aimed to determine novel potential prognostic biomarkers shared between primary lung squamous cell tumors (without lymph node metastasis) and lymphatic metastasis, using the Cancer Genome Atlas database. Differentially expressed genes were screened by limma statistical package in R environment. Gene ontology and biological pathways analyses were performed using Enrichr for up-regulated and down-regulated genes. Also, we selected lymph node metastasis related genes among DEGs using correlation analysis between DEGs and suitable references genes for metastasis. Receiver operating characteristic curves was applied using pROC and R package ggplot2 to evaluate diagnostic value of differentially expressed genes. In addition, survival and drug resistance analyses were performed for differentially expressed genes. The miRNA-mRNA interaction networks were predicted by miRwalk and TargetScan databases and expression levels analysis of the miRNAs which were mainly targeting mRNAs was performed using UALCAN database. Protein–protein interaction network analysis and hub genes identification were performed using FunRich and Cytoscape plugin cytoHubba. In this study, a total of 397 genes were differentially expressed not only with a significant difference between N + vs. normal and N0 vs. normal but also with significant difference between N + vs. N0. Identified GO terms and biological pathways were consistent with DEGs role in the lung squamous cell carcinoma and lymph node metastasis. A significant correlation between 56 genes out of 397 differentially expressed genes with reference genes prompted them being considered for identifying lymph node metastasis of lung squamous cell carcinoma. In addition, SLC46A2, ZNF367, AC107214.1 and NCBP1 genes were identified as survival-related genes of patients with lung squamous cell carcinoma. Moreover, NEDD9, MRPL21, SNRPF, and SCLT1 genes were identified to be involved in lung squamous cell carcinoma drug sensitivity/resistance. We have identified several numbers of miRNAs and their related target genes which could emerge as potential diagnostic biomarkers. Finally, CDK1, PLK1, PCNA, ZWINT and NDC80 identified as hub genes for underlying molecular mechanisms of lung squamous cell carcinoma and lymphatic metastasis. Our study highlights new target genes according to their relation to lymph node metastasis, whose expressions in the primary lung squamous cell carcinoma are able to accurately assess the presence of lymphatic metastasis.

The Role of the Matrix Metalloproteinase-9 Gene in Tumor Development and Metastasis: A Narrative Review

Matrix metalloproteinase-9 (MMP-9) is one of the widely studied enzymes of the extracellular matrix which can degrade various matrix biomolecules. The gene coding for this enzyme has been found to be associated with various multifactorial diseases, including cancer. More specifically, the expression of MMP-9 and polymorphisms of its gene have been found to be correlated with the formation and the invasiveness of different types of cancer. Hence, the latter gene can potentially be used both as a clinical genetic marker and a possible target in anticancer therapy. The present minireview explores the role of the MMP-9 gene in the process of tumor formation, growth, and metastasis and presents an overview of the polymorphisms of the gene associated with cancer as well as its regulation mechanisms, to provide an insight into the potential clinical applications. Nevertheless, further clinical trials and research are still required to reach more valuable conclusions for the clinical implications of the recent findings.

Abstract 4997: PLK1 promotes the metastasis and drug resistance in melanoma

Abstract Melanoma is one of the most frequently diagnosed cancers in the caucasian population of both genders. The survival rate of advanced melanoma patients will be exceedingly poor, as low as 27.3%. BRAF-V600E is the most common mutation found clinically, resulting in the constitutive activation of the MAPK signaling pathway. Vemurafenib, a specific inhibitor of mutant BRAF, has shown an impressive response in phase 3 clinical trials and was approved by FDA in 2011. However, due to the rapid development of resistance, the duration of response under a single treatment is frequently short, highlighting the urgent requirement for novel therapy in melanoma treatment. Polo-like kinase 1 (PLK1), a crucial cell cycle regulator, participates in multiple mitotic processes, including centrosome maturation, mitotic entry, spindle assembly, sister chromatid segregation, mitotic exit, and cytokinesis. Compared to normal tissues, the expression level of PLK1 significantly upregulates in multiple cancers. More importantly, the expression level of PLK1 negatively correlates with the melanoma patients’ survival rate based on the TCGA database. Besides, PLK1 is identified as an oncogene to promote proliferation, motility, and resistance to various drugs. In the present study, we found that overexpression of PLK1 promotes tumor growth, metastasis, metabolic reprogramming, and vemurafenib resistance in the BrafCA/Ptenloxp mouse melanoma model. Through phosphorylation, overexpressed PLK1 stabilizes BACH1, a critical transcriptional factor of multiple metastatic and metabolic genes, and protects it from degradation. Subsequently, a higher level of BACH1 leads to increasing metastasis, upregulation of glycolysis, and suppression of OXPHOs. Besides, we have validated the strong synergy between PLK1 inhibitor volasertib and vemurafenib in treating melanoma carrying BRAF-V600E. Our in vitro and in vivo experiments have shown an improved efficacy of this combined therapy on inhibition of cell proliferation, induction of cell death, and suppression of cell metastasis compared to mono-treatment. In short, our data has indicated PLK1 as a potent and promising target in cancer treatment. Citation Format: Fengyi Mao, Yifan Kong, Chaohao Li, Yanquan Zhang, Xiongjian Rao, Qiongsi Zhang, Xinyi Wang, Zhiguo Li, Xiaoqi Liu. PLK1 promotes the metastasis and drug resistance in melanoma. [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 4997.