Multi-ancestral origin of intestinal tumors: Impact on growth, progression, and drug efficacy.

BACKGROUND: Data are steadily accruing that demonstrate that intestinal tumors are frequently derived from multiple founding cells, resulting in tumors comprised of distinct ancestral clones that might cooperate or alternatively compete, thereby potentially impacting different phases of the disease process. AIM: We sought to determine whether tumors with a multi-ancestral architecture involving at least two distinct clones show increased tumor number, growth, progression, or resistance to drug intervention. METHODS: Mice carrying the Min allele of Apc were generated that were mosaic with only a subset of cells in the intestinal epithelium expressing an activated form of PI3K, a key regulatory kinase affecting several important cellular processes. These cells were identifiable as they fluoresced green, whereas all other cells fluoresced red. RESULTS: Cell lineage tracing revealed that many intestinal tumors from our mouse model were derived from at least two founding cells, those expressing the activated PI3K (green) and those which did not (red). Heterotypic tumors with a multi-ancestral architecture as evidenced by a mixture of green and red cells exhibited increased tumor growth and invasiveness. Clonal architecture also had an impact on tumor response to low-dose aspirin. Aspirin treatment resulted in a greater reduction of heterotypic tumors derived from multiple founding cells as compared to tumors derived from a single founding cell. CONCLUSION: These data indicate that genetically distinct tumor-founding cells can contribute to early intratumoral heterogeneity. The coevolution of the founding cells and their progeny enhances colon tumor progression and impacts the response to aspirin. These findings are important to a more complete understanding of tumorigenesis with consequences for several distinct models of tumor evolution. They also have practical implications to the clinic. Mouse models with heterogenous tumors are likely better for predicting drug efficacy as compared to models in which the tumors are highly homogeneous. Moreover, understanding how interactions among different populations in a single heterotypic tumor with a multi-ancestral architecture impact response to a single agent and combination therapies are necessary to fully develop personalized medicine.

A Computational-Based Approach to Identify Estrogen Receptor α/ß Heterodimer Selective Ligands.

The biologic effects of estrogens are transduced by two estrogen receptors (ERs), ERα and ERß, which function in dimer forms. The ERα/α homodimer promotes and the ERß/ß inhibits estrogen-dependent growth of mammary epithelial cells; the functions of ERα/ß heterodimers remain elusive. Using compounds that promote ERα/ß heterodimerization, we have previously shown that ERα/ß heterodimers appeared to inhibit tumor cell growth and migration in vitro. Further dissection of ERα/ß heterodimer functions was hampered by the lack of ERα/ß heterodimer-specific ligands. Herein, we report a multistep workflow to identify the selective ERα/ß heterodimer-inducing compound. Phytoestrogenic compounds were first screened for ER transcriptional activity using reporter assays and ER dimerization preference using a bioluminescence resonance energy transfer assay. The top hits were subjected to in silico modeling to identify the pharmacophore that confers ERα/ß heterodimer specificity. The pharmacophore encompassing seven features that are potentially important for the formation of the ERα/ß heterodimer was retrieved and subsequently used for virtual screening of large chemical libraries. Four chemical compounds were identified that selectively induce ERα/ß heterodimers over their respective homodimers. Such ligands will become unique tools to reveal the functional insights of ERα/ß heterodimers.

New insights into the earliest stages of colorectal tumorigenesis.

INTRODUCTION: Tumors in the large intestine have been postulated to arise via a stepwise accumulation of mutations, a process that takes up to 20 years. Recent advances in lineage tracing and DNA sequencing, however, are revealing new evolutionary models that better explain the vast amount of heterogeneity observed within and across colorectal tumors. Areas covered: A review of the literature supporting a novel model of colorectal tumor evolution was conducted. The following commentary examines the basic science and clinical evidence supporting a modified view of tumor initiation and progression in the colon. Expert commentary: The proposed 'cancer punctuated equilibrium' model of tumor evolution better explains the variability seen within and across polyps of the colon and rectum. Small colorectal polyps (6-9mm) followed longitudinally by interval imaging with CT colonography have been reported to have multiple fates: some growing, some remaining static in size, and others regressing in size over time. This new model allows for this variability in growth behavior and supports the hypothesis that some tumors can be 'born to be bad' as originally postulated by Sottoriva and colleagues, with very early molecular events impacting tumor fitness and growth behavior in the later stages of the disease process.

Subclonal diversity arises early even in small colorectal tumours and contributes to differential growth fates.

OBJECTIVE AND DESIGN: The goal of the study was to determine whether the mutational profile of early colorectal polyps correlated with growth behaviour. The growth of small polyps (6-9 mm) that were first identified during routine screening of patients was monitored over time by interval imaging with CT colonography. Mutations in these lesions with known growth rates were identified by targeted next-generation sequencing. The timing of mutational events was estimated using computer modelling and statistical inference considering several parameters including allele frequency and fitness. RESULTS: The mutational landscape of small polyps is varied both within individual polyps and among the group as a whole but no single alteration was correlated with growth behaviour. Polyps carried 0-3 pathogenic mutations with the most frequent being in APC, KRAS/NRAS, BRAF, FBXW7 and TP53. In polyps with two or more pathogenic mutations, allele frequencies were often variable, indicating the presence of multiple populations within a single tumour. Based on computer modelling, detectable mutations occurred at a mean polyp size of 30±35 crypts, well before the tumour is of a clinically detectable size. CONCLUSIONS: These data indicate that small colon polyps can have multiple pathogenic mutations in crucial driver genes that arise early in the existence of a tumour. Understanding the molecular pathway of tumourigenesis and clonal evolution in polyps that are at risk for progressing to invasive cancers will allow us to begin to better predict which polyps are more likely to progress into adenocarcinomas and which patients are at greater risk of developing advanced disease.

The Multidisciplinary Management of Colorectal Cancer: Present and Future Paradigms.

As treatment strategies for patients with colorectal cancer advance, there has now become an ever-increasing need for multidisciplinary teams to care for these patients. Recent investigations into the timing and duration of perioperative therapy, as well as, the rise of molecular profiling have led to more systemic chemotherapeutic options. The most efficacious use, in terms of timing and patient selection, of these therapies in the setting of modern operative and radiotherapy techniques requires the generation of care teams discussing cases at multidisciplinary conferences. This review highlights the role of multidisciplinary team conferences, advances in perioperative chemotherapy, current clinical biomarkers, and emerging therapeutic agents for molecular subtypes of metastatic colon cancer. As our understanding of relevant molecular subtypes increases and as data becomes available on treatment response, the treatment of colorectal cancer will become more precise and effective.

Understanding Intratumoral Heterogeneity: Lessons from the Analysis of At-Risk Tissue and Premalignant Lesions in the Colon.

Advances in DNA sequencing have created new opportunities to better understand the biology of cancers. Attention is currently focused on precision medicine: does a cancer carry a mutation that is targetable with already available drugs? But, the timing at which multiple, targetable mutations arise during the adenoma to carcinoma sequence remains unresolved. Borras and colleagues identified mutations and allelic imbalance in at-risk mucosa and early polyps in the human colon. Their analyses indicate that mutations in key genes can arise quite early during tumorigenesis and that polyps are often multiclonal with at least two clones. These results are consistent with the "Big Bang" model of tumorigenesis, which postulates that intratumoral heterogeneity is a consequence of a mutational burst in the first few cell divisions following initiation that drives divergence from a single founder with unique but related clones coevolving. Emerging questions center around the ancestry of the tumor and impact of early intratumoral heterogeneity on tumor establishment, growth, progression, and most importantly, response to therapeutic intervention. Additional sequencing studies in which samples, especially at-risk tissue and premalignant neoplasms, are analyzed from animal models and humans will further our understanding of tumorigenesis and lead to more effective strategies for prevention and treatment. Cancer Prev Res; 9(8); 638-41. ©2016 AACRSee related article by Borras, et al., Cancer Prev Res 2016;9(6):417-427.

Molecular Profiles Guide Colorectal Cancer Treatment.

An improved understanding of colorectal cancer as a collection of multiple cancer subtypes is paving the way to precision medicine-based treatments.

Improved Detection of Microsatellite Instability in Early Colorectal Lesions.

Microsatellite instability (MSI) occurs in over 90% of Lynch syndrome cancers and is considered a hallmark of the disease. MSI is an early event in colon tumor development, but screening polyps for MSI remains controversial because of reduced sensitivity compared to more advanced neoplasms. To increase sensitivity, we investigated the use of a novel type of marker consisting of long mononucleotide repeat (LMR) tracts. Adenomas from 160 patients, ranging in age from 29-55 years old, were screened for MSI using the new markers and compared with current marker panels and immunohistochemistry standards. Overall, 15 tumors were scored as MSI-High using the LMRs compared to 9 for the NCI panel and 8 for the MSI Analysis System (Promega). This difference represents at least a 1.7-fold increase in detection of MSI-High lesions over currently available markers. Moreover, the number of MSI-positive markers per sample and the size of allelic changes were significantly greater with the LMRs (p = 0.001), which increased confidence in MSI classification. The overall sensitivity and specificity of the LMR panel for detection of mismatch repair deficient lesions were 100% and 96%, respectively. In comparison, the sensitivity and specificity of the MSI Analysis System were 67% and 100%; and for the NCI panel, 75% and 97%. The difference in sensitivity between the LMR panel and the other panels was statistically significant (p<0.001). The increased sensitivity for detection of MSI-High phenotype in early colorectal lesions with the new LMR markers indicates that MSI screening for the early detection of Lynch syndrome might be feasible.

Dynamic tumor growth patterns in a novel murine model of colorectal cancer.

Colorectal cancer often arises from adenomatous colonic polyps. Polyps can grow and progress to cancer, but may also remain static in size, regress, or resolve. Predicting which polyps progress and which remain benign is difficult. We developed a novel long-lived murine model of colorectal cancer with tumors that can be followed by colonoscopy. Our aim was to assess whether these tumors have similar growth patterns and histologic fates to human colorectal polyps to identify features to aid in risk stratification of colonic tumors. Long-lived Apc(Min/+) mice were treated with dextran sodium sulfate to promote colonic tumorigenesis. Tumor growth patterns were characterized by serial colonoscopy with biopsies obtained for immunohistochemistry and gene expression profiling. Tumors grew, remained static, regressed, or resolved over time with different relative frequencies. Newly developed tumors demonstrated higher rates of growth and resolution than more established tumors that tended to remain static in size. Colonic tumors were hyperplastic lesions (3%), adenomas (73%), intramucosal carcinomas (20%), or adenocarcinomas (3%). Interestingly, the level of ß-catenin was higher in adenomas that became intratumoral carcinomas than those that failed to progress. In addition, differentially expressed genes between adenomas and intramucosal carcinomas were identified. This novel murine model of intestinal tumorigenesis develops colonic tumors that can be monitored by serial colonoscopy, mirror growth patterns seen in human colorectal polyps, and progress to colorectal cancer. Further characterization of cellular and molecular features is needed to determine which features can be used to risk-stratify polyps for progression to colorectal cancer and potentially guide prevention strategies.

Differential action of monohydroxylated polycyclic aromatic hydrocarbons with estrogen receptors α and ß.

Polycyclic aromatic hydrocarbons (PAHs) are a diverse group of widespread environmental pollutants, some of which have been found to be estrogenic or antiestrogenic. Recent data have shown that hydroxylated PAH metabolites may be responsible for the estrogenic effects of some PAHs. The purpose of this study was to investigate the effects of several PAHs, as well as their monohydroxylated metabolites, on estrogen receptors (ERs), ERα and ERß. Three parent PAHs and their monohydroxylated metabolites were each evaluated using transcriptional reporter assays in isogenic stable cell lines to measure receptor activation, competitive binding assays to determine ligand binding, and bioluminescence resonance energy transfer assays to assess dimerization. Finally, the estrogenic effects of the hydroxylated metabolites were confirmed by quantitative real-time PCR of estrogen-responsive target genes. Although the parent PAHs did not induce ERα or ERß transcriptional activity, all of the monohydroxylated PAHs (1-OH naphthanol, 9-OH phenanthrene, 1-OH pyrene) selectively induced ERß transcriptional activity at the concentrations tested, while not activating ERα. Additionally, the monohydroxylated PAHs were able to competively bind ERß, induce ERß homodimers, and regulate ERß target genes. Although monohydroxylated PAHs appeared to have weak agonist activity to ERß, our results showed that they can elicit a biologically active response from ERß in human breast cancer cells and potentially interfere with ERß signaling pathways.