Molecular drivers of tumor progression in microsatellite stable APC mutation-negative colorectal cancers

The tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut–) CRCs. We analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut– and APC mutation-positive (APCmut+) microsatellite stable CRCs. Transcriptionally, APCmut– CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut– CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3. APCmut– CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

Molecular Drivers of Tumor Progression in Microsatellite Stable APC Mutation-Negative Colorectal Cancers

ABSTRACTBackgroundThe tumor suppressor gene adenomatous polyposis coli (APC) is the initiating mutation in approximately 80% of all colorectal cancers (CRC), underscoring the importance of aberrant regulation of intracellular WNT signaling in CRC development. Recent studies have found that early-onset CRC exhibits an increased proportion of tumors lacking an APC mutation. We set out to identify mechanisms underlying APC mutation-negative (APCmut–) CRCs.MethodsWe analyzed data from The Cancer Genome Atlas to compare clinical phenotypes, somatic mutations, copy number variations, gene fusions, RNA expression, and DNA methylation profiles between APCmut– and APC mutation-positive (APCmut+) microsatellite stable CRCs.ResultsTranscriptionally, APCmut– CRCs clustered into two approximately equal groups. Cluster One was associated with enhanced mitochondrial activation. Cluster Two was strikingly associated with genetic inactivation or decreased RNA expression of the WNT antagonist RNF43, increased expression of the WNT agonist RSPO3, activating mutation of BRAF, or increased methylation and decreased expression of AXIN2. APCmut– CRCs exhibited evidence of increased immune cell infiltration, with significant correlation between M2 macrophages and RSPO3.ConclusionsAPCmut– CRCs comprise two groups of tumors characterized by enhanced mitochondrial activation or increased sensitivity to extracellular WNT, suggesting that they could be respectively susceptible to inhibition of these pathways.

A Case of Adult Pancreatoblastoma With Novel APC Mutation and Genetic Heterogeneity

BackgroundPancreatoblastoma is a rare malignant epithelial neoplasm of the pancreas that mainly occurs in children and involves abnormalities in the WNT/β-catenin pathway, such as CTNNB1 mutation. However, the molecular abnormalities in adult pancreatoblastoma are not well known.Case PresentationAn elderly man, who underwent elective distal pancreatectomy and splenectomy, was referred to our hospital with a mass in the tail of the pancreas. Histologically, the lesion revealed proliferation of clear, basophilic, and cartilaginous tumor cells with lymphatic metastasis. Each of the morphologically distinct tumor components showed different immunohistochemical patterns, indicating heterogeneous differentiation, including epithelial (both acinar and ductal), mesenchymal, and neuroendocrine differentiation. All tumor components showed nuclear expression of β-catenin and cyclin D1. Per next-generation sequencing (NGS), the clear and basophilic tumor cells shared mutations in APC, GRM8, LAMP1, and AKA9. Among the mutations, APC, c.1816_1817insA showed the highest frequency in both cell types, indicating that APC mutation was a driver mutation of the tumor. A diagnosis of PB was rendered.SummaryIn conclusion, the clear and basophilic cells of the tumor were supposedly derived from the same clone and subsequently acquired additional mutations. This is the first report of clonal evolution in pancreatoblastoma.

BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer

To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9’s role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints

Genetics in Colorectal Cancer

Colorectal cancer are the third most commonly diagnosed form of cancer globally, which about 11% of all cancer diagnoses. Obesity, together with kind of sedentary lifestyle, red meat consumption, tobacco, and alcohol consumption are considered as predisposing factors of progression of CRC. In the development of colorectal cancer, genetic factors having a role in its incidence. The hereditary type of colorectal cancer was divided into two types is polyposis and Lynch syndrome which have each and different mechanism and genetic pattern. Lynch syndrome contributes for 3–5% of CRC cases and is caused by a germline mutation in one of four genes associated with the DNA mismatch repair (MMR) system: MLH1, MSH2, MSH6, or PMS2. In polyposis, there are some types such as Familial Adenomatous Polyposis (FAP) which mostly caused by APC mutation, MYH-associated polyposis, and the rare hamartomatous polyposis syndromes. Genetic testing and better family history documentation can enable those with a hereditary predisposition for the neoplasm to take preventive measures. Some genetic testing can be used such as Microsatellite instability (MSI), immunohistochemistry (IHC), DNA sequencing and protein truncation that used for each type of hereditary colorectal cancer.

ErbB Receptor Stimulation Is Required for Mouse Colon Adenoma Organoids to Form Crypts

The majority of colon adenomas harbor genetic mutations in the APC gene. APC mutation leads to changes in Wnt signalling and cell-cell adhesion: as a consequence, intestinal crypt budding increases and the excess crypts accumulate to form adenomas that progress to colon cancer. When cultured with Wnt, R-spondin, EGF, Noggin, myofibroblast conditioned medium and Matrigel, crypts from normal mouse colon mucosa form crypt-producing organoids and can be passaged continuously. Under the same culture and passage conditions, crypts isolated from colon adenomas derived from Apcmin/+ mice typically grow as spheroidal cysts and do not produce crypts. The adenoma organoid growth requires EGF, but not Wnt, R-spondin or Noggin. However, when mouse colon adenoma spheroids are grown for more than 10 days in the presence of EGF, crypt formation occurs. EGF, EREG, β-cellulin, Neuregulin-1 or AREG are sufficient for initiating crypt formation, however, neuregulin-1 is more potent than the other EGF-family members. EGFR and ErbB2 inhibitors both prevent crypt formation in adenoma cultures. Either EGFR:ErbB2 or ErbB3:ErbB2 signalling is sufficient to initiate adenoma crypt budding and elongation. ErbB2 inhibitors may provide a therapeutic avenue for controlling and ablating colon adenomas.

Reconstruction of Contemporary Human Stem Cell Dynamics with Oscillatory Molecular Clocks

Molecular clocks record cellular ancestry. However, currently used clocks 'tick too slowly' to measure the short-timescale dynamics of cellular renewal in adult tissues. Here we develop 'rapidly oscillating DNA methylation clocks' where ongoing (de)methylation causes the clock to 'tick-tock' back-and-forth between methylated and unmethylated states like a pendulum. We identify oscillators using standard methylation arrays and develop a mathematical modelling framework to quantitatively measure human adult stem cell dynamics from these data. Small intestinal crypts were inferred to contain slightly more stem cells than colon (6.5 ± 1.0 vs 5.8 ± 1.7 stem cells/crypt) with slower stem cell replacement in small intestine (0.79 ± 0.5 vs 1.1 ± 0.8 replacements/stem cell/year). Germline APC mutation increased the number of replacements per crypt (13.0 ± 2.4 replacements/crypt/year vs 6.9 ± 4.6 for healthy colon). In blood, we measure rapid expansion of acute leukaemia and slower growth of chronic disease. Rapidly oscillating molecular clocks are a new methodology to quantitatively measure human somatic cell dynamics.

Heterozygous APC germline mutations impart predisposition to colorectal cancer

Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.