Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer.

BACKGROUND: Recently, consensus molecular subtypes (CMSs) have been proposed as a robust transcriptome-based classification system for colorectal cancer (CRC). Tetraspanins (TSPANs) are transmembrane proteins. They have been associated with the development of numerous malignancies, including CRC, through their role as "master organizers" for multi-molecular membrane complexes. No previous study has investigated the correlation between TSPANs and CMS classification. Herein, we investigated the expression of TSPANs in patient-derived primary CRC tissues and their CMS classifications. METHODS: RNA samples were derived from primary CRC tissues (n = 100 patients diagnosed with colorectal adenocarcinoma) and subjected to RNA sequencing for transcriptome-based CMS classification and TSPAN-relevant analyses. Immunohistochemistry (IHC) and immunofluorescence (IF) stains were conducted to observe the protein expression level. To evaluate the relative biological pathways, gene-set enrichment analysis was performed. RESULTS: Of the highly expressed TSPAN genes in CRC tissues (TSPAN8, TSPAN29, and TSPAN30), TSPAN8 was notably overexpressed in CMS3-classified primary tissues. The overexpression of TSPAN8 protein in CMS3 CRC was also observed by IHC and IF staining. As a result of gene-set enrichment analysis, TSPAN8 may potentially play a role in organizing signaling complexes for kinase-based metabolic deregulation in CMS3 CRC. CONCLUSIONS: The present study reports the overexpression of TSPAN8 in CMS3 CRC. This study proposes TSPAN8 as a subtype-specific biomarker for CMS3 CRC. This finding provides a foundation for future CMS-based studies of CRC, a complex disease and the second leading cause of cancer mortality worldwide.

Spatial Transcriptomic Profiling of Tetraspanins in Stage 4 Colon Cancer from Primary Tumor and Liver Metastasis.

Stage 4 colon cancer (CC) presents a significant global health challenge due to its poor prognosis and limited treatment options. Tetraspanins, the transmembrane proteins involved in crucial cancer processes, have recently gained attention as diagnostic markers and therapeutic targets. However, their spatial expression and potential roles in stage 4 CC tissues remain unknown. Using the GeoMx digital spatial profiler, we profiled all 33 human tetraspanin genes in 48 areas within stage 4 CC tissues, segmented into immune, fibroblast, and tumor compartments. Our results unveiled diverse gene expression patterns across different primary tumor sub-regions. CD53 exhibited distinct overexpression in the immune compartment, hinting at a potential role in immune modulation. TSPAN9 was specifically overexpressed in the fibroblast compartment, suggesting involvement in tumor invasion and metastasis. CD9, CD151, TSPAN1, TSPAN3, TSPAN8, and TSPAN13 displayed specific overexpression in the tumor compartment, indicating potential roles in tumor growth. Furthermore, our differential analysis revealed significant spatial changes in tetraspanin expression between patient-matched stage 4 primary CC and metastatic liver tissues. These findings provide spatially resolved insights into the expression and potential roles of tetraspanins in stage 4 CC progression, proposing their utility as diagnostic markers and therapeutic targets. Understanding this landscape is beneficial for tailoring therapeutic strategies to specific sub-tumor regions in the context of stage 4 CC and liver metastasis.

Exploring extracellular matrix and prostaglandin pathway alterations across varying resection margin distances of right-sided colonic adenocarcinoma.

BACKGROUND: Surgical resection followed by indicated adjuvant therapy offers potential curative treatment in colonic adenocarcinoma. Beyond the well-established seed and soil theory of colon cancer progression, the 'normal-appearing' tissues near the tumor are not genuinely normal and remain as remnants in patients following surgery. Our objective was to elucidate the alteration of gene expression and pathways across various distances of resection margins in right-sided colonic adenocarcinoma. METHODS: Twenty-seven fresh samples of primary cancer and 56 matched non-tumor tissues adjacent to the tumor (NAT) were collected from patients with resectable right-sided colon cancer. NAT were systematically obtained at varying distances (1, 5, and 10 cm) on both proximal and distal sides. Comprehensive gene expression analysis was performed using 770-gene PanCancer Progression Panel, delineating distinctive pathways and functional predictions for each region. RESULTS: Distinctive gene signatures and pathways exhibited by normal-appearing tissues were discovered at varying distances from cancer. Notably, SFRP2, PTGDS, COL1A1, IL1B, THBS2, PTGIS, COL1A2, NPR1, and BGN were upregulated, while ENPEP, MMP1, and NRCAM were downregulated significantly in 1-cm tissue compared to farther distances. Substantial alterations in the extracellular matrix (ECM) and prostaglandin/thromboxane synthesis were significantly evident at the 1-cm distance. Functional analysis indicated enhanced cell viability and survival, alongside reduced cellular death and apoptosis. CONCLUSIONS: Different distances exerted a significant impact on gene alteration within the normal-looking mucosa surrounding primary cancer, influenced by various mechanisms. These findings may highlight potential therapeutic targets related to the ECM and prostaglandin/thromboxane pathways for treatment strategies.

The KRAS-Mutant Consensus Molecular Subtype 3 Reveals an Immunosuppressive Tumor Microenvironment in Colorectal Cancer.

Colorectal cancers (CRC) with KRAS mutations (KRASmut) are frequently included in consensus molecular subtype 3 (CMS3) with profound metabolic deregulation. We explored the transcriptomic impact of KRASmut, focusing on the tumor microenvironment (TME) and pathways beyond metabolic deregulation. The status of KRASmut in patients with CRC was investigated and overall survival (OS) was compared with wild-type KRAS (KRASwt). Next, we identified CMS, and further investigated differentially expressed genes (DEG) of KRASmut and distinctive pathways. Lastly, we used spatially resolved gene expression profiling to define the effect of KRASmut in the TME regions of CMS3-classified CRC tissues. CRC patients with KRASmut were mainly enriched in CMS3. Their specific enrichments of immune gene signatures in immunosuppressive TME were associated with worse OS. Activation of TGFß signaling by KRASmut was related to reduced pro-inflammatory and cytokine gene signatures, leading to suppression of immune infiltration. Digital spatial profiling in TME regions of KRASmut CMS3-classified tissues suggested up-regulated genes, CD40, CTLA4, ARG1, STAT3, IDO, and CD274, that could be characteristic of immune suppression in TME. This study may help to depict the complex transcriptomic profile of KRASmut in immunosuppressive TME. Future studies and clinical trials in CRC patients with KRASmut should consider these transcriptional landscapes.

Impact of Washing Processes on RNA Quantity and Quality in Patient-Derived Colorectal Cancer Tissues.

Background: Colorectal cancer (CRC) is a common and lethal cancer worldwide. Extraction of high-quality RNA from CRC samples plays a key role in scientific research and translational medicine. Specimen collection and washing methods that do not compromise RNA quality or quantity are needed to ensure high quality specimens for gene expression analysis and other RNA-based downstream applications. We investigated the effect of tissue specimen collection and different preparation processes on the quality and quantity of RNA extracted from surgical CRC tissues. Materials and Methods: After surgical resection, tissues were harvested and prepared with various washing processes in a room adjacent to the operating room. One hundred fourteen tissues from 36 CRC patients were separately washed in either cold phosphate-buffered saline reagent (n = 34) or Dulbecco's modified Eagle's medium (DMEM; n = 34) for 2-3 minutes until the stool was removed, and unwashed specimens served as controls (n = 34). Six tissue specimens were washed and immersed in DMEM for up to 1 hour at 4°C. Before RNA extraction, all specimens were kept in the stabilizing reagent for 3 months at -80°C. RNA was extracted, and the concentration per milligram of tissue was measured. RNA quality was assessed using the RNA integrity number (RIN) value. Results: Different washing processes did not result in significant differences in RNA quantity or RIN values. In the six tissues that were washed and immersed in DMEM for 1 hour, RIN values significantly decreased. The quality of the extracted RNA from most specimens was excellent with the average RIN greater than 7. Conclusions: RNA is stable in specimens washed in different processes for short periods, but RIN values may decrease with prolonged wash times.

Risks and cancer associations of metachronous and synchronous multiple primary cancers: a 25-year retrospective study.

BACKGROUND: The situation of patients developing multiple primary cancers is becoming more frequent and graver. This study investigated the risks of developing second primary cancers that are related to first primary cancers, and the interval times of synchronous and metachronous multiple primary cancers. PATIENTS AND METHODS: Retrospective data were retrieved from 109,054 patients aged ≥18 who were diagnosed with a first solid cancer and registered at Siriraj Cancer Center between 1991 and 2015. A two-month period between first- and second- primary cancers was used to differentiate metachronous and synchronous multiple primary cancers. The combinations of subsequent cancers and relative risks (RRs) of having multiple primary cancers versus having single primary cancer for the top-ten first and second primary cancers were examined. The RR was adjusted for age of the first primary cancer. A survival analysis of the time to second-primary-cancer development was performed. RESULTS: Multiple primary cancers were found in 1785 (1.63%) patients. Most (70.87%) second primary cancers occurred after 2 months of first breast, skin, colorectal, lung, head and neck, liver, male genital cancer-prostate, thyroid, and female genital cancer-non-uterine cancers, resulting in those cancers being classified as metachronous multiple primary cancer. After adjustment for age at first diagnosis, head and neck cancers had the highest metachronous association with second esophageal cancers (RR, 25.06; 95% CI, 13.41-50.77). Prostate cancer and second colorectal cancer also demonstrated a high metachronous association (RR, 2.00; 95% CI, 1.25-3.05). A strong synchronous association was found between uterine and ovarian cancers (RR, 27.77; 95% CI, 17.97-43.63). The median time from the first uterine cancer to second-cancer development was 55 days. CONCLUSIONS: The top-ten most frequent multiple primary cancers were the following: breast; liver; head and neck; colorectal; male genital cancer-prostate; skin; female genital cancer-uterine; thyroid; lung; and female genital cancer-non-uterine. Second primary cancers showed specific associations that depended on the first primary cancer. Physicians should be cognizant of the most common combinations and the interval times of metachronous and synchronous multiple primary cancers.

Genistein reduces the activation of AKT and EGFR, and the production of IL6 in cholangiocarcinoma cells involving estrogen and estrogen receptors.

Cholangiocarcinoma (CCA) is a malignant tumor of the biliary epithelium associated with Opisthorchis viverrini, primary sclerosing cholangitis and hepatitis viral infection. In the global population, men have higher incidence rates for CCA than women; thus, a gender disparity in the progression of chronic inflammation of the biliary duct leading to malignancy may involve the effects of estrogen (E2). Genistein (GE), a prominent phytoestrogen found in soy products, is an estrogen receptor ß (ERß) agonist and a tyrosine kinase inhibitor. The present study investigated the effects of GE on the growth of CCA cells by cell viability assay. The effects on signaling proteins were detected by western blot analysis and ELISA. Gene expression was examined by RT-qPCR. Two human intrahepatic CCA cell lines, HuCCA­1 and RMCCA­1, were utilized. GE (50­200 µM) reduced the viability of the two cell lines, and also inhibited the activation of epidermal growth factor receptor (EGFR) and AKT, as evidenced by decreasing protein levels of phosphorylated (p)-EGFR (Tyr1173) and p­AKT (Ser473), respectively. GE altered the mitogen­activated protein kinase signaling cascade by mediating decreased protein levels of p­p38 and increased protein levels of p­ERK1/2. GE significantly decreased the levels of interleukin 6 (IL6) and induced the expression of inducible nitric oxide synthase (iNOS). GE also downregulated the expression of p­ERα (Ser118) protein and ERα mRNA levels. Finally, GE induced the downregulation of the protein levels of ERß. Of note, E2 deprivation potentiated the GE-induced reduction of p­EGFR (Tyr1173) and total AKT proteins and production of IL6, and mediated the downregulation of GE-induced iNOS protein. In conclusion, GE inhibited the growth of human CCA cell lines by reducing the activation of EGFR and AKT, and by attenuating the production of IL6. E2 and ER were also involved in the growth-inhibitory effect of GE in CCA cells.