Aneuploid Colorectal Cancers Research Articles

Spatial UBE2N protein expression indicates genomic instability in colorectal cancers

BackgroundOne major hallmark of colorectal cancers (CRC) is genomic instability with its contribution to tumor heterogeneity and therapy resistance. To facilitate the investigation of intra-sample phenotypes and the de novo identification of tumor sub-populations, imaging mass spectrometry (IMS) provides a powerful technique to elucidate the spatial distribution patterns of peptides and proteins in tissue sections.MethodsIn the present study, we analyzed an in-house compiled tissue microarray (n = 60) comprising CRCs and control tissues by IMS. After obtaining protein profiles through direct analysis of tissue sections, two validation sets were used for immunohistochemical evaluation.ResultsA total of 28 m/z values in the mass range 800–3500 Da distinguished euploid from aneuploid CRCs (p < 0.001, ROC AUC values < 0.385 or > 0.635). After liquid chromatograph-mass spectrometry identification, UBE2N could be successfully validated by immunohistochemistry in the initial sample cohort (p = 0.0274, ROC AUC = 0.7937) and in an independent sample set of 90 clinical specimens (p = 0.0070, ROC AUC = 0.6957).ConclusionsThe results showed that FFPE protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value for improved molecular classification. Particularly, the protein expression of UBE2N was validated in an independent clinical cohort to distinguish euploid from aneuploid CRCs.

Aneuploid Cell Survival Relies upon Sphingolipid Homeostasis.

Aneuploidy, a hallmark of cancer cells, poses an appealing opportunity for cancer treatment and prevention strategies. Using a cell-based screen to identify small molecules that could selectively kill aneuploid cells, we identified the compound N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide monohydrochloride (DL-PDMP), an antagonist of UDP-glucose ceramide glucosyltransferase. DL-PDMP selectively inhibited proliferation of aneuploid primary mouse embryonic fibroblasts and aneuploid colorectal cancer cells. Its selective cytotoxic effects were based on further accentuating the elevated levels of ceramide, which characterize aneuploid cells, leading to increased apoptosis. We observed that DL-PDMP could also enhance the cytotoxic effects of paclitaxel, a standard-of-care chemotherapeutic agent that causes aneuploidy, in human colon cancer and mouse lymphoma cells. Our results offer pharmacologic evidence that the aneuploid state in cancer cells can be targeted selectively for therapeutic purposes, or for reducing the toxicity of taxane-based drug regimens. Cancer Res; 77(19); 5272-86. ©2017 AACR.

Abstract 3584: Genomic analysis reveals a role for BCL9L in aneuploidy tolerance in colorectal cancer

Abstract Cancer genomes are frequently characterized by changes in the number and structure of the normal diploid karyotype, normally denominated as chromosomal instability (CIN), that result in tumour cell populations with high karyotipic diversity. CIN plays a key role in tumour evolution by contributing to intratumour heterogeneity and is often associated with key clinical landmarks such as bad prognosis and drug resistance. CIN originates from deficient segregation of chromosomes during anaphase, which generates sister cells with abnormal karyotypes. Additionally, mechanisms of tolerance need to be acquired by cancer cells to survive segregation errors. In colorectal cancer (CRC), 20% of tumours present high levels of microsatellite instability (MSI) together with low levels of CIN. The remaining 80% present low or no MSI, together with a wide range of CIN. In this project, we explored the mutational landscape of 25 MSI-negative CRC samples in order to extract somatic mutational patterns of aneuploid CRC associated with CIN tolerance or chromosomal missgregation. Notably, we found frequent truncating mutations in BCL9L and loss-of-heterozigosity in 5 aneuploid samples. Further inspection of somatic alterations in the TCGA cohort showed BCL9L alterations in 14% of CIN CRC (mutations and deletions) and more importantly, bioinformatic analysis demonstrated that these tumours have significantly higher wGII scores (the fraction of the genome with alterations). We also compared wGII scores of samples with co-occurring BCL9L and TP53 mutations and found that tumours with alterations in both genes show higher levels of CIN than those with only one alteration. Functional characterization of BCL9L showed that depletion in various diploid cell lines induces tolerance of drug-induced and naturally occurring segregation errors. Long-term BCL9L-depleted cells increased the fraction of aneuploid cells in a diploid cell line and also, and enhancement of engraftment efficiency when injected into immune deficient mice. Additionally, CRISPR mediated inactivation of BCL9L showed enrichment in monoallelic BCL9L mutant colonies when treated with a drug that induces segregation errora, which supports a role for BCL9L haploinsufficiency in CIN tolerance. Finally, we observed that BCL9L regulates the expression of Caspase-2, a proteolytic enzyme that is activated in a p53-independent manner by segregation errors and cleaves several proteins important for cell survival such as the p53 regulator MDM2 and the mitochondrial apoptotic regulator protein BID. Citation Format: Carlos Lopez-Garcia, Nicholas McGranahan, Sebastjian Hobor, Nicolai Birkbak, Enric Domingo, Andrew Rowan, Ian Tomlinson, Charles Swanton. Genomic analysis reveals a role for BCL9L in aneuploidy tolerance in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3584.

HDAC2 and TXNL1 distinguish aneuploid from diploid colorectal cancers

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Further understanding of the translation of DNA aneuploidy into protein expression will help to define novel biomarkers to improve therapies and prognosis. DNA ploidy was assessed by image cytometry. Comparison of gel-electrophoresis-based protein expression patterns of three diploid and four aneuploid colorectal cancer cell lines detected 64 ploidy-associated proteins. Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in two overlapping high-ranked networks maintaining Cellular Assembly and Organization, Cell Cycle, and Cellular Growth and Proliferation. CAPZA1, TXNL1, and HDAC2 were significantly validated by Western blotting in cell lines and the latter two showed expression differences also in clinical samples using a tissue microarray of normal mucosa (n=19), diploid (n=31), and aneuploid (n=47) carcinomas. The results suggest that distinct protein expression patterns, affecting TXNL1 and HDAC2, distinguish aneuploid with poor prognosis from diploid colorectal cancers.

Nucleation capacity and presence of centrioles define a distinct category of centrosome abnormalities that induces multipolar mitoses in cancer cells

Analysis of centrosome number and structure has become one means of assessing the potential for aberrant chromosome segregation and aneuploidy in tumor cells. Centrosome amplification directly causes multipolar catastrophic mitoses in mouse embryonic fibroblasts (MEFs) deficient for the tumor suppressor genes Brca1 or Trp53. We observed supernumerary centrosomes in cell lines established from aneuploid, but not from diploid, colorectal carcinomas; however, multipolar mitoses were never observed. This discrepancy prompted us to thoroughly characterize the centrosome abnormalities in these and other cancer cell lines with respect to both structure and function. The most striking result was that supernumerary centrosomes in aneuploid colorectal cancer cell lines were unable to nucleate microtubules, despite the presence of gamma-tubulin, pericentrin, PLK1, and AURKA. Analysis by scanning electron microscopy revealed that these supernumerary structures are devoid of centrioles, a result significantly different from observations in aneuploid pancreatic cancer cell lines and in Trp53 or Brca1 deficient MEFs. Thus, multipolar mitoses are dependent upon the ability of extra gamma-tubulin containing structures to nucleate microtubules, and this correlated with the presence of centrioles. The assessment of centrosome function with respect to chromosome segregation must therefore take into consideration the presence of centrioles and the capacity to nucleate microtubules. The patterns and mechanisms of chromosomal aberrations in hematologic malignancies and solid tumors are fundamentally different. The former is characterized by specific chromosome translocations, whose consequence is the activation of oncogenes. Most carcinomas, however, reveal variations in the nuclear DNA content. The observed genomic imbalances and gross variations in chromosome number can result from unequal chromosome segregation during mitotic cell division. It is therefore fundamental to elucidate mechanisms involved in distribution of the genome to daughter cells. Prior to cell division, the centrosome organizes microtubules and the mitotic spindle. Deciphering the consequences of alterations in centrosome number, structure, and function is an important step towards understanding how a diploid genome is maintained. Although extra centrosomes have now been observed in carcinomas and were correlated with aneuploidy, a careful functional investigation of these structures and their role in generating chromosome imbalances may lead to the identification of distinct mechanistic pathways of genomic instability. Understanding these pathways will also be important in determining whether they are potential molecular targets of therapeutic intervention.

DNA Ploidy Status and Prognosis in Colorectal Cancer: A Meta-Analysis of Published Data

In colorectal cancer, the negative effect of aneuploidy has been a controversy for more than 20 years. Studies to determine a survival-deoxyribonucleic acid content relationship have conflicting results. A systematic literature search followed by a meta-analysis of published studies addressing prognostic effect of aneuploidy for patients who underwent surgical treatment of colon and rectal cancer was conducted. The main outcome measure was the five-year overall mortality rate after surgical resection. For the selected studies, we estimated this outcome for three subsets of patients through separate meta-analyses: 1) for all patients with colorectal cancer; 2) only between patients with Stage II colon cancer; and 3) only for studies in which follow-up losses were declared. The presence of publication bias was assessed with a funnel plot for asymmetry. A total of 5,478 patients with colorectal cancer were represented in 32 studies (Group 1), we estimated a reduction in the five-year overall mortality from 43.2 percent for aneuploid tumors to 29.2 percent for diploid tumors (combined relative risk = 1.44; 95 percent confidence interval = 1.34-1.55; P < 0.001). In addition, 357 patients with Stage II colon cancer (Group 2) extracted from three studies had an absolute reduction of 14.3 percent in five-year overall mortality favoring diploid tumors (combined relative risk = 1.93; 95 percent confidence interval = 1.29-2.89; P = 0.001). Lastly, of 14 studies in which follow-up losses were declared (Group 3), 2,221 patients were represented and a 15.7 percent mortality reduction was measured favoring patients with diploid tumors (combined relative risk = 1.44; 95 percent confidence interval = 1.3-1.61; P < 0.001). Patients who undergo an aneuploid colorectal cancer surgical resection have a higher risk of death after five years. This finding may ultimately impact survival of patients with node-negative colon cancer through adjuvant therapy.

Analysis of subclonal expansion of colorectal carcinomas by flow cytometry.

DNA heterogeneity of colorectal carcinomas has been investigated by flow cytometry, most studies have focused on the clinical usefulness of DNA ploidy analysis. Since cancers consist of predominant subclones with proliferative advantage due to clonal expansion, we attempted to analyse the clonal expansion of colorectal carcinomas within a tumour by measuring DNA ploidy. The DNA ploidy and heterogeneity of multiple fresh samples obtained from 164 colorectal adenocarcinomas were analysed by flow cytometry. Each tumour was divided into an average of six specimens, which were analysed separately. For 146 of the tumours (89%) at least one DNA aneuploid population was found within the cancer tissue examined. DNA multiploidy was detected in 26 cases (17.8%) among the cancers with aneuploidy. Based on the DNA index (DI), hypertriploid aneuploidy (1.7<DI<1.8) was found most frequently in the aneuploid colorectal cancers examined. DNA ploidy heterogeneity was seen in 75 (51.4%) of the DNA aneuploid tumours. There were only 3 cases with more than three subclones including a diploid line. The present results indicate that colorectal carcinomas consist of a few dominant subclones and have a DNA content (hypertriploid aneuploid) that confers a proliferative advantage.

Clinical significance of the DNA index in hematogenous dissemination of aneuploid colorectal cancer.

The DNA index (DI), defined as the ratio of the G0/G1 channel number of tumor cells to the G0/G1 channel number of stromal cells, was analyzed in 121 aneuploid colorectal cancers. In patients with aneuploid tumors, a high DI significantly correlated with tumor penetration beyond the proper muscle layer, positive vascular invasion, and the presence of liver metastasis. This observation led us to conclude that a high DI is likely to be associated with hematogenous dissemination in aneuploid colorectal cancer.

Tumor DNA Content in Resectable, Primary Colorectal Carcinoma

Tumor DNA content was measured in patients with colorectal carcinoma in order to determine whether tumor ploidy was a prognostic indicator independent of standard clinical and pathologic characteristics. One hundred forty-seven patients were analyzed who had their primary resectable colorectal carcinomas resected with curative intent from 1974 to 1981. Aneuploid colorectal cancers (i.e., tumors with abnormal DNA content) tended to be less well-differentiated, to invade the serosa or extend beyond, and to have lymph node metastases rather than diploid tumors (i.e., tumors with normal DNA content). A significantly increased rate of recurrent disease was demonstrated in patients with aneuploid tumors as opposed to those with diploid tumors (46.7% vs. 4.8%, respectively [p less than 0.001]). In addition, patients with aneuploid tumors exhibited a significantly decreased disease-free and overall survival in comparison with patients with diploid colorectal carcinomas. A Cox regression analysis demonstrated that tumor DNA content was the single most important factor in predicting recurrence or death from colorectal carcinoma.