Awad Jarrar

Epigenomic Enhancer Profiling Defines a Signature of Colon Cancer

Colorectal Cancer Signature The mutations and genome aberrations that characterize cancer result in often dramatically altered gene and protein expression patterns. It is these altered expression patterns that directly and indirectly drive progression of the disease. In human primary colorectal cancer cells, Akhtar-Zaidi et al. (p. 736, published online 12 April) analyzed the pattern of epigenetically modified chromatin at “enhancer” sequences that are known to be critical in the control of gene expression. An epigenetic enhancer signature was defined that was specifically associated with colorectal cancer cells. Methylation tags at long-distance gene regulatory elements provide a signature specific to cancer cells. Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A

Chemotherapy stimulates cancer-associated fibroblasts to secrete interleukin-17A to provide maintenance cues to support the growth of colorectal cancer-initiating cells.

Defining Phenotypes and Cancer Risk in Hyperplastic Polyposis Syndrome

BACKGROUND: Hyperplastic polyposis syndrome is a rare syndrome of colorectal cancer predisposition. Patterns of inheritance of hyperplastic polyposis syndrome are not obvious and the clinical definition is relatively arbitrary. We hypothesize that there are multiple phenotypes included in what is currently called hyperplastic polyposis syndrome. We performed this review of a large series of patients who presented with multiple serrated polyps to look for clinical patterns that may confirm our hypothesis. METHODS: Hereditary colorectal cancer, colonoscopy, and clinical databases from a single institution were queried for patients meeting the following criteria: 1) ≥20 serrated colorectal polyps; 2) ≥5 serrated polyps proximal to the sigmoid; 3) ≥2 serrated polyps ≥10 mm in size; 4) any serrated polyps in a person with at least one first-degree relative who has hyperplastic polyposis syndrome. Records were reviewed for demographics, polyp details, and personal or family history of colorectal extracolonic malignancy. RESULTS: One-hundred fifteen patients were included. Median age at diagnosis was 62 years and 56% were male. Ninety-seven percent were white. Twenty-five percent of patients had a personal history and 38% had a family history of colorectal cancer. Twenty-eight percent of patients had a personal history and 54% had a family history of extracolonic cancer. Phenotype analysis identified 3 patterns: relatively few large, right-sided polyps (n = 55), many small left-sided polyps (n = 18), and a combination of both left- and right-sided polyps (n = 42). The right-sided phenotype had more sessile serrated polyps and tended to develop colorectal cancer at a younger age. CONCLUSIONS: There are at least 3 different but overlapping clinical phenotypes within hyperplastic polyposis. Recognizing this clinical heterogeneity is important in defining underlying genetic causes.

BRAF mutations in colorectal cancer are associated with distinct clinical characteristics and worse prognosis.

BACKGROUND: Colorectal cancer is a heterogeneous disease with multiple underlying genetic mutations causing different clinical phenotypes. Mutation in the BRAF oncogene is a key step in malignant transformation within the methylator pathway to colorectal cancer. However, there is a paucity of information about BRAF mutant colorectal tumors. OBJECTIVE: This study defines the clinical characteristics and oncologic outcome associated with colorectal cancer BRAF mutations. DESIGN: Colorectal adenocarcinomas from a single-institution frozen-tumor biobank were studied. Genomic DNA was isolated and analyzed for mutations in the BRAF oncogene by polymerase chain reaction amplification followed by direct sequencing. A sample was classified as mutant if any of the tested loci were mutated. Patient and tumor characteristics were recorded including patient age, sex, tumor location, tumor differentiation, and microsatellite instability. MAIN OUTCOME MEASURES: Statistical associations with BRAF mutant tumors were determined by the Fisher exact probability test, &khgr;2 test, or Wilcoxon analysis. Kaplan-Meier estimates and multivariate Cox regression analysis were performed for overall survival. RESULTS: Four hundred seventy-five colorectal adenocarcinomas were included in the study population; 56 samples harbored a BRAF mutation (12%). There were significant differences between BRAF wild-type and mutant tumors in age (66 vs 75 years, p = 0.004), female sex (44% vs 71%, p < 0.001), proximal tumor location (44% vs 95%, p < 0.001), and frequency of microsatellite instability (16% vs 76%, p < 0.001). There was no difference in cancer stage between BRAF mutant and wild-type populations. Survival data were analyzed for 322 patients with stage I to III disease, and patients with a BRAF mutation had decreased overall survival than those without a mutation (p = 0.018). With the use of Cox regression analysis, BRAF mutation conferred a worse overall survival (HR 1.79, CI 1.05–3.05, p = 0.03) independent of microsatellite instability status. CONCLUSIONS: BRAF mutations in colorectal cancers are associated with distinct clinical characteristics and worse prognosis.

Advancement flap repair: a good option for complex anorectal fistulas.

BACKGROUND: Rectal advancement flap is a popular option for treatment of complex anal fistula. Although early outcomes vary, concerns remain about postoperative continence, long-term healing, and its role in patients with Crohns disease and anovaginal fistulas. PURPOSE: This study aimed to report long-term outcomes in patients with complex fistula disease. PATIENTS: Patients who were undergoing rectal advancement flap for anal fistula were included in the study. DESIGN: Patients were contacted to determine the status of their fistula disease, their bowel function, and their degree of fecal incontinence. MAIN OUTCOME MEASURES: The main outcomes measured were healing rate and continence. RESULTS: There were 98 patients, 43 men and 55 women, mean age 53 ± 14 years. Fifty-eight had cryptoglandular fistulas, and 40 (41%) had IBD (33 had Crohns disease). Seventy-seven of 98 patients had perianal fistulas, and all 77 underwent seton drainage before advancement flap. Twenty-one women had anovaginal fistulas. Average postoperative length of stay was 3 ± 1 days. There were no mortalities. Follow-up was possible in 75 patients, a mean of 7 ± 3 years after surgery. Primary healing occurred in 54 (72%) patients. Twenty-one patients (28%) underwent further treatment, and 12 (57%) healed after a second advancement flap. Four more patients healed after more than 2 flaps or fistulotomy leading to an overall healing rate of 70 of 75 (93%). Patients with Crohns disease had lower healing rates than those with cryptoglandular fistulas (87% vs 98%). Thirty-two patients (43%) had normal fecal continence before flap, and 43 (57%) had normal fecal continence after flap. CONCLUSION: Advancement flap is a good option for patients with complex anal fistulas.

Screening for Thyroid Cancer in Patients With Familial Adenomatous Polyposis

Objective:Clarify the incidence of thyroid cancer in patients with Familial adenomatous polyposis (FAP) in a prospective study of thyroid neck US screening. Background:FAP is a hereditary disease predisposing to cancer in multiple organs, including the thyroid. However, routine thyroid screening for FAP patients is not generally practiced in the United States. Here, we report the initial results of a prospective thyroid cancer screening program in patients with FAP. Methods:At the time of yearly gastrointestinal follow-up, every FAP patient in our registry was offered thyroid ultrasound (US) performed by experienced endocrine surgeons. Clinical findings related to thyroid disease were analyzed for those patients who completed screening from August 2008 to December 2009. Results:Of 192 screened FAP patients, 72 (38%) had thyroid nodules and 5 (2.6%) had thyroid cancer. Three of 5 patients with FAP and thyroid cancer were women. Four of 5 patients had the multifocal papillary type with mean size 15 mm. Clinical history and neck exam did not detect any of the 5 cancers. Conclusion:The incidence of thyroid cancer among FAP patients is high. Medical history and exam are inadequate to identify patients with thyroid cancer, thus thyroid screening with US is warranted.

Differential Connexin Function Enhances Self-Renewal in Glioblastoma

SUMMARY The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self‐renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self‐renewal and tumor initiation. To overcome these barriers in triple‐negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well‐established pluripotency gene NANOG. NANOG‐GFP+ cells gave rise to both GFP+ and GFP− cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self‐renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well‐established CSC markers CD24−/CD44+, CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG‐GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule‐A (JAM‐A). JAM‐A was highly expressed in GFP+ cells and patient‐derived xenograft ALDH+ CSCs compared with the GFP− and ALDH− cells, respectively. Depletion of JAM‐A compromised self‐renewal, whereas JAM‐A overexpression induced self‐renewal in GFP− cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non‐CSCs in TNBC that can be used to identify CSC‐specific targets for the development of future therapeutic strategies. Stem Cells. Stem Cells 2015;33:2114–2125

Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor‐mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid‐derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune‐suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self‐renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5‐flurouracil (5‐FU) in a low‐dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient‐derived CSCs but not nonstem tumor cells selectively drove MDSC‐mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune‐suppressive enzyme arginase‐1 in MDSCs in a CXCR2‐dependent manner, whereas blocking MIF reduced arginase‐1 production. Similarly to 5‐FU, targeting tumor‐derived MIF conferred a survival advantage to tumor‐bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self‐renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026–2039

Multiplex Flow Cytometry Barcoding and Antibody Arrays Identify Surface Antigen Profiles of Primary and Metastatic Colon Cancer Cell Lines

Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.