Introduction

Ranking 3^rd in the most diagnosed cancer and 2^nd in the leading cause of cancer deaths globally, colorectal carcinoma (CRC) is characterized by the nuanced multi-step carcinogenesis in which it acquires the hallmarks of cancer. Metastatic CRC (mCRC), which majorly appears in an advanced stage, has only a 12% 5-year survival. Notably, immediate resection of mCRC is rare, and the lack of operability motivates the further uncovering of carcinogenic mechanisms to broaden our therapeutic options.
Our group has directed our focus on alternative splicing event which is frequently reported to be hijacked in cancer. By long-read sequencing in CRC organoids, we identified a novel isoform of citrate synthase (CS) with unprecedented exon-skipping (ΔCS) apart from the full-length isoform (CS-WT). Being the rate-limiting enzyme of the tricarboxylic acid cycle, differentially expressed ΔCS in CRC might be explanatory to metabolic reprogramming phenotype and may serve as a potential target of therapeutic intervention.

Aims & Methods

We validated the sequencing results in normal colonic (n=5), primary CRC (pCRC, n=10), and mCRC organoids (n=3), and in-house patient tumor and adjacent normal tissue pairs (n=97) by RT-qPCR. We performed survival analyses with progression-free survival, disease-free survival, and overall survival using all stages, late-stage, and early-stage stratification to explore the clinical relevance of ΔCS. We then proceeded into in vitro experimental validation in CRC cell lines, and in vivo tail-vein injection in NSG mice for investigating the biological phenomena. We also inspected the molecular mechanism to explain the oncogenic phenomena by focusing on the potential structural effects and enzymatic activity upon exon-skipping in ΔCS.

Results

An in-frame spliced CS transcript unreported in RefSeq and GENECODE was found upregulated in patient-derived pCRC organoids over normal organoids, and mCRC organoids have the highest ΔCS expression among the 3 groups. The clinical relevance of ΔCS was proven by 97 pairs of human CRC tissues, demonstrating a significant overexpression over non-tumoral colonic tissues (Paired student’s t-test, p<0.001). Tumor-to-adjacent normal (T/AN) ratio analysis indicated that 80.4% of patients showed increased expression of ΔCS in tumor tissues. A significant association was observed between high ΔCS expression and recurrence (Fisher’s exact test, p<0.01). Kaplan-Meier analyses, stratified by median expression, were performed and the high ΔCS expression group was found associated with lower survival rates when compared to the low ΔCS group in both PFS (Log-rank test, p<0.01) and DFS (p<0.005). We further separated groups by cancer stages and found that high ΔCS expression leads to worse survival outcomes in stage III & IV patients, indicating the metastatic feature associated with this isoform (OS: p<0.005; PFS: p<0.005).
Moreover, ΔCS silencing (shΔCS) demonstrated a more profound inhibition of cell proliferation compared with CS-WT knock-down (shCS-WT) and control. shΔCS groups also showed a significant increase in apoptosis cell counts. Overexpression of ΔCS showed enhanced migration ability in vitro and in vivo. The dimerization effect by ΔCS leads to enhanced CS activity, and metabolic shift to amino acids synthesis was found by GC-MS profiling.

Conclusion

Unannotated ΔCS isoform is explanatory to clinically important metastatic phenotype in CRC through metabolic alteration.

Introduction

Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality worldwide. A remarkable increase in early onset colorectal cancer (EOCRC), defined as diagnosis before the age of 50 years, has been observed globally over the past decades. Recent data indicate that EOCRC presents distinct clinicopathological features, such as advanced stage at diagnosis and unique molecular signatures. Interestingly, EOCRC has been reported to be more likely to occur in the left sided colon, commonly called early onset left sided colorectal cancer (EOLCC). However, the underlying molecular mechanisms of EOLCC carcinogenesis and progression have still not been fully explored.
Concurrently, the prevalence of metabolic syndrome (MetS), including central obesity, insulin resistance, hypertension, and dyslipidemia, has increased in younger populations. As reported, MetS promotes carcinogenesis through chronic inflammation, hyperinsulinemia, and oxidative stress. Importantly, MetS is more common in patients with EOLCC. Although a hypothesis has been proposed regarding the possible link between MetS and EOLCC, the genetic and molecular mechanisms involved remain largely unexplored. Moreover, possible targeted treatment strategies for patients with both conditions remain limited.

Aims & Methods

This study aimed to investigate and elucidate key biomarkers associated with MetS-related EOLCC progression and treatment response.
The in-hospital cohort was employed to evaluate the clinical implications of the original tumor location in early-onset colorectal cancer (EOCRC). By utilizing differentially expressed genes (DEGs) and weighted gene coexpression network analysis (WGCNA), we identified potential genes associated with MetS-related EOLCC. The potential mechanisms of MetS-related EOLCC were analyzed using functional pathway enrichment. Random forest (RF) and support vector machine recursive feature elimination (SVM-RFE) algorithms were used to screen candidate biomarkers. The analyses of survival implications, expression patterns, and diagnostic performance were conducted to identify the key biomarkers. The responses to treatment were evaluated. Potential candidate compounds were screened, and molecular docking was performed. The expression and functional signatures were validated in single-cell RNA sequencing (scRNA-seq) datasets and experiments in vitro.

Results

The in-hospital cohort revealed the EOCRC patients had the greater proportion of EOLCC. Through the use of the edgdR package and WGCNA, we identified co-expressed genes common to both EOLCC and MetS, which are notably enriched in pathways related to stromal remodeling and metabolic regulation. By utilizing machine learning, 3 candidate biomarkers were highlighted. Moreover, only CD151 was associated to the prognosis of EOLCC and advanced stage. CD151 were found closely linked to stromal reconstruction and chemoresistance. Meanwhile, the potential compounds targeted to MetS-related EOLCC were identified by molecular docking. Subsequent scRNA-seq analysis confirmed the expression and function patterns of CD151, with especially prominent expression in tumor cells. Ultimately, the bioinformatics findings were validated by quantitative real-time PCR (qRT-PCR) and Immunohistochemical (IHC) staining of clinical samples.

Conclusion

This study investigated CD151 as the key biomarker in MetS-related EOLCC, leading to insights into prognosis, tumor biological status, and personalized treatment strategies. This key biomarker serves as a valuable reference for further exploring the development of clinical applications.

References

1. Wolf AMD, Fontham ETH, Church TR, et al. (2018) Colorectal cancer screening for average-risk adults: 2018 guideline update from the American Cancer Society. CA Cancer J Clin 68: 250–281.
2. Frostberg E, Rahr HB (2020) Clinical characteristics and a rising incidence of early-onset colorectal cancer in a nationwide cohort of 521 patients aged 18-40 years. Cancer Epidemiol 66: 101704.
3. Soto J, Gutiérrez Alonso N, Bringas Beranek M, et al. (2022) Analysis of survival trends, clinical, and molecular characteristics of patients with early-onset colorectal cancer (EOCRC). J Clin Oncol 40: 59–59.
4. Dharwadkar P, Greenan G, Singal AG, et al. (2021) Is Colorectal Cancer in Patients Younger Than 50 Years of Age the Same Disease as in Older Patients? Clin Gastroenterol Hepatol Off Clin Pract J Am Gastroenterol Assoc 19: 192-194.e3.
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Introduction

As common precursors to colorectal cancer (CRC), polyps and adenomas in the large intestine may develop into malignancy over time through a cascade known as the "normal-polyp-adenoma-carcinoma" sequence. Colorectal cancer (CRC) is the third most common cancer and the leading cause of cancer-related death worldwide^​, with an estimated increase to 3.2 million new cases and 1.6 million deaths by 2040^​. The high mortality of CRC is largely due to the late diagnosis, uncomplete molecular understanding of pathological progression, and limited treatment options for patients with advanced disease stage. Therefore, the identification of novel biomarkers at early (pre-cancerous) phase, thorough elucidation of pathological mechanisms, and development of effective therapies is crucial for CRC diagnosis and treatment.

Aims & Methods

Here we have utilized 10× genomics scRNA-seq platform to perform a high-resolution transcriptomic analysis of three normal colorectal tissues, two samples with inflammatory polyps, two specimens with adenomatous polyps and three CRC samples.

Results

By analyzing the scRNA-seq data of the ten human colorectal samples, which represent the four phases of colorectal pathogenesis, in combination with validation by immunofluorescence staining using expanded clinical samples, and together with verification using large scale TCGA and Kaplan–Meier data, we revealed a dynamic molecular landscape and for the first time demonstrated the importance of ligand-receptor genes, transcription factors and genes encoding multiple immunoglobulins and heat shock proteins driving the colorectal carcinogenesis.
Besides the heterogeneity of (sub-) cell types and difference of the transcriptomic profiling across the four stages, one of the most interesting and novel findings of this study is that the expression of ligand-receptor genes can be used to determine the cell types and even cell sub-types, at least for the colorectal tissues collected in this study, not the expressions of other gene sets, such as cell surface receptor signaling pathways, cell-cell signaling, transcription factors, cytokines, cell activation factors, cell signaling factors, or extracellular matrisome, implying that ligand-receptor pairs are fundamentally important for the cell type determination and cell-cell communications in colorectal tissues.
We found that some genes encoding immunoglobulin members are highly expressed in the T cells (IGHA1, IGHA2, IGKC and IGLC2) and in the fibroblasts (IGHA1, IGHA2, IGLC1, IGLC2, IGKC and JCHAIN) from normal colorectal tissues, while some genes encoding heat shock proteins are highly expressed in T cells (HSP90AA1, HSP90AB1, HSPA1B, HSPD1, HSPE1 and HSPH1) and plasma cells (HSP90AA1, HSPA1A, HSPA1B, HSPA6, HSPB1 and HSPD1) from abnormal colorectal samples, especially in the ones with adenomatous polyps.
Furthermore, IgG plasma cells can be grouped into two sub-types, characteristic of high expression of immunoglobulin-encoding genes IGKC and IGLC1/IGLC2, respectively. In addition, the six clusters of IgA plasma cells can also be characterized based on the combinatory expression level of some immunoglobulin-encoding genes (IGHA1, IGHA2, IGKC, IGLC1, IGLC2 and IGLC3). In summary, our analyses suggested the potential distinct functional importance of immunoglobulins and heat shock proteins in immunity and microenvironment during the colorectal malignancy.

Conclusion

Our transcriptome data provides valuable insights and a useful resource for better understanding the molecular pathological landscape in CRC to achieve more effective diagnosis and treatments.

References

Siegel, R.L., Wagle, N.S., Cercek, A., Smith, R.A., and Jemal, A. (2023). Colorectal cancer statistics, 2023. CA Cancer J Clin 73, 233-254. 10.3322/caac.21772.
2. Morgan, E., Arnold, M., Gini, A., Lorenzoni, V., Cabasag, C.J., Laversanne, M., Vignat, J., Ferlay, J., Murphy, N., and Bray, F. (2023). Global burden of colorectal cancer in 2020 and 2040: incidence and mortality estimates from GLOBOCAN. Gut 72, 338-344. 10.1136/gutjnl-2022-327736.
3. Huck, M.B., and Bohl, J.L. (2016). Colonic Polyps: Diagnosis and Surveillance. Clin Colon Rectal Surg 29, 296-305. 10.1055/s-0036-1584091.
4. He, D.G., Chen, X.J., Huang, J.N., Chen, J.G., Lv, M.Y., Huang, T.Z., Lan, P., and He, X.S. (2022). Increased risk of colorectal neoplasia in inflammatory bowel disease patients with post-inflammatory polyps: A systematic review and meta-analysis. World J Gastrointest Oncol 14, 348-361. 10.4251/wjgo.v14.i1.348.
5. Rudy, D.R., and Zdon, M.J. (2000). Update on colorectal cancer. Am Fam Physician 61, 1759-1770, 1773-1754.
6. Heumos, L., Schaar, A.C., Lance, C., Litinetskaya, A., Drost, F., Zappia, L., Lucken, M.D., Strobl, D.C., Henao, J., Curion, F., et al. (2023). Best practices for single-cell analysis across modalities. Nat Rev Genet 24, 550-572. 10.1038/s41576-023-00586-w.

Introduction

Colorectal cancer (CRC) is rising among young adults. It is cancer of colon, appendix and rectum. According to GLOBOCON 2018, It is the 4^th most common cancer in the world and the third leading cause of cancer mortality. The expected rise of the CRC rate to 60% by year of 2030 globally, will result in 2.2 million new cases and 1.1 million deaths annually. CRC is Heterogeneous,70% cases of CRCs are sporadic in onset, 10-30% are familial and 5-7% are due to hereditary diseases.

Aims & Methods

To evaluate the clinicopathological features of Colorectal Cancer (CRC) among young adults under the age of 30).
This was cross-sectional Retrospective study conducted at the Gastroenterology and Oncology Department of JPMC, Karachi from Jan. 2018 till Dec. 2022. All patients with CRC under age 30 were included in the study. Diagnosis was made on Colonoscopy and histopathology confirming CRC.The data obtained was analyzed on the statistical software SPSS version 23. Descriptive statistics were obtained by frequencies and percentages.

Results

In this study total 566 patients of CRC and Young patients 117 were analyzed. The majority, 67 (57.3%) patients were male and 50 (42.7%) were female. Most commonly present in Urdu speaking 47(40%) followed by Sindhi 40 (34%) and Pathan and Panjabi 13(11%). Smoking has a strong association,13(9%) were active smokers followed by Naswar 8 (6%).Most common area was from Orangi Town 8(6%) followed by Korangi 3(4%) Colonoscopy revealed polypoidal growth in 79 (67%) of patients followed by ulcerated lesions in 23 (19.7%). The most common site of CRC was Rectum affecting 48 (41%) followed by Sigmoid colon 46 (39.3%) and the least common site was Ascending colon 7(2.6%). Moderately differentiated Adenocarcinoma was the most common histological type found in 96 (82.5%) and 17 (10%) having signet ring type. The common clinical presentation was abdominal pain 64 (53%) constipation 45 (38.5%) and Bleeding per Rectum 42 (36%).

Conclusion

CRC is the leading cause of mortality. It is on rising pattern among young adults, most commonly involving the left side of the colon. We need more studies to make guidelines for screening and surveillance of CRC in our population.