Stepwise Accumulation Of Genetic Alterations Research Articles

Mucin phenotype and genetic alterations in non-V600E BRAF-mutated colorectal cancer

Colorectal cancer (CRC) is a heterogeneous disease that develops through stepwise accumulation of genetic alterations and progresses via several distinct pathways. However, the tumorigenesis of CRCs with BRAF non-V600E mutations remains unclear. Here, we aimed to elucidate the tumorigenesis of CRCs with BRAF non-V600E mutations, focusing on differences in mucin phenotype and genetic alterations between CRCs with non-V600E and V600E mutations. We investigated 201 patients with CRC and performed panel testing of 415 genes to identify BRAF mutations. Patients were classified into five mucin phenotypes – large-intestinal, small-intestinal, gastric, mixed, and unclassified – using immunohistochemistry for CD10, MUC2, MUC5AC, and MUC6. BRAF mutations were identified in 24 of 201 patients’ samples, of which 13 (6.5%) had a V600E mutation (V600E-mutant) and 11 (5.5%) had non-V600E mutations (non-V600E-mutant). MUC5AC expression was significantly associated with V600E mutations (P = 0.040), while CD10 expression was significantly associated with non-V600E mutations (P = 0.010). The small-intestinal mucin phenotype was significantly associated with non-V600E mutations (P = 0.031), while the mixed mucin phenotype was significantly associated with V600E mutations (P = 0.027). Regarding genetic alterations, focusing on the WNT signaling pathway, APC mutation was significantly associated with non-V600E mutations (P < 0.001), while RNF43 mutation was significantly associated with V600E mutations (P = 0.020). Considering the differences in mucin phenotype and genetic alterations, different modes of tumorigenesis are assumed for CRC with BRAF V600E mutation and non-V600E mutations. These findings are important in understanding the biology and treatment strategies for BRAF-mutant CRC.

Abstract 165: Establishment and characterization of Vogelgram-mimic colorectal cancer organoids as a laboratory tool

Abstract Colorectal cancer is a deadly disease with a 14% five-year survival rate once it metastasizes. The development of colorectal cancer involves a stepwise accumulation of genetic alterations in APC, KRAS, DCC, and p53 that is often described as a “Vogelgram,” named after the Vogelstein laboratory which laid the groundwork for our understanding of the molecular events leading to colorectal cancer. Organoids are a highly physiologically relevant laboratory tool that can provide valuable information regarding the tumor microenvironment. Here, we describe ex vivo establishment of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl colorectal organoids, which behave normally before tamoxifen treatment but acquire malignant behaviors after tamoxifen-induced loss of APC and p53. Colonic crypts from mice with colon-specific expression of APC fl/fl, CRE+, KRAS G12D +/-, P53 fl/fl were isolated and grown in Matrigel and WENRAS medium following the Organoid Core Facility protocol of the Legorreta Cancer Center. Western blot was used to measure levels of KRAS, KRAS G12D, APC, and p53 after 12 days of tamoxifen treatment and images were taken after 6 days of tamoxifen treatment. The organoid growth medium was collected after 3-4 days of incubation and run on the Luminex 200 platform to measure the levels of 50+ cytokines. Organoids were embedded in OCT and cryosectioned for IF staining with p53, p21, Ki67 and Lgr5 antibodies. IF stained organoid slides were imaged by confocal microscope at 40X magnification. Tamoxifen treatment (12 days) induces overexpression of KRAD G12D protein, reduces expression of APC, and abolishes p53 expression. At day 6, distinct morphological changes indicate malignant transformation. This transformation was accompanied by a trend toward decreased BAFF and CCL20. H&amp;E staining revealed clearly visible intra-organoid structures and florescent labeling of Ki67, Lgr5, and p21 visualized by confocal microscope revealed distinct localization of these proteins within the organoid structures. Here, we describe Vogelgram-mimicking organoids that may be used as a valuable laboratory tool to study the processes involved in the stepwise transformation into colorectal cancer. We plan to use this validated system to evaluate drug sensitivity, cytokine secretion, and immune cell interactions along colorectal cancer progression. We also plan to characterize cell populations with single-cell RNA-seq, establish triangulated cultures, and conduct in vivo studies using this transgenic mouse model. Citation Format: Leiqing Zhang, Lindsey Carlsen, Lanlan Zhou, Liz Hernandez Borrero, Wafik S. El-Deiry. Establishment and characterization of Vogelgram-mimic colorectal cancer organoids as a laboratory tool [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 165.

A Dynamic Transcription Factor Signature Along the Colorectal Adenoma-Carcinoma Sequence in Patients With Co-Occurrent Adenoma and Carcinoma.

BackgroundColorectal carcinoma (CRC) often arises from benign adenoma after a stepwise accumulation of genetic alterations. Here, we profiled the dynamic landscapes of transcription factors (TFs) in the mucosa-adenoma-carcinoma progression sequence.MethodsThe transcriptome data of co-occurrent adenoma, carcinoma, and normal mucosa samples were obtained from GSE117606. Identification of differentially expressed TFs (DE-TFs) and subsequent function annotation were conducted in R software. Expression patterns of DE-TFs were clustered by Short Time-series Expression Miner software. Thereafter, modular co-expression analysis, Kaplan-Meier survival analysis, mutation profiling, and gene set enrichment analysis were conducted to investigate TF dynamics in colorectal tumorigenesis. Finally, tissue microarrays, including 51 tumors, 32 adenomas, and 53 normal tissues, were employed to examine the expression of significant candidates by immunohistochemistry staining.ResultsCompared to normal tissues, 20 (in adenoma samples) and 29 (in tumor samples) DE-TFs were identified. During the disease course, 28 expression patterns for DE-TFs and four co-expression modules were clustered. Notably, six DE-TFs, DACH1, GTF2IRD1, MEIS2, NR3C2, SOX9, and SPIB, were identified as having a dynamic signature along the colorectal adenoma-carcinoma sequence. The dynamic signature was of significance in GO enrichment, prognosis, and co-expression analysis. Among the 6-TF signature, the roles of GTF2IRD1, SPIB and NR3C2 in CRC progression are unclear. Immunohistochemistry validation showed that GTF2IRD1 enhanced significantly throughout the mucosa-adenoma-carcinoma sequence, while SPIB and NR3C2 kept decreasing in stroma during the disease course.ConclusionsOur study provided a dynamic 6-TF signature throughout the course of colorectal mucosa-adenoma-carcinoma. These findings deepened the understanding of colorectal cancer pathogenesis.

Po-327 Modelling intratumor heterogeneity through CRISPR-barcodes

IntroductionCancer is an evolutionary process in which the stepwise accumulation of genetic alterations is shaped by Darwinian selection. As a result, each tumour is composed of a complex mixture of clonal cell subpopulations containing a partially overlapping, but distinct, pattern of driver and passenger mutations. Such intratumor heterogeneity has dramatic consequences, not only for cancer progression and metastatic spread, but also for resistance to therapy. We recently devised a novel approach based on CRISPR/Cas9 technology to recapitulate and trace the emergence of a new mutation in a subset of cancer cells, thus enabling functional studies on a gene of interest in a context that mimics intratumor heterogeneity.Material and methodsWe used CRISPR/Cas9 technology to insert a potentially functional modification in the sequence of a gene of interest, coupled to a series of silent point mutations, serving as a genetic label for cell tracing. Because of HDR low efficiency, the modified allele can be introduced only in a small fraction of cells, thus mimicking the emergence of new mutant clones within a cancer mass population. In parallel, a second barcode, consisting of different silent mutations, was used as an internal control for possible CRISPR off-target cleavage. After exposing the cells to a given selective condition, the effects of the mutation of interest can be analysed by measuring the proportion of each barcode in genomic DNA by real-time quantitative PCR or deep sequencing.Results and discussionsWe used this CRISPR-barcoding strategy to model different mechanisms of non-small-cell lung cancer resistance to EGFR inhibitors, and we established a multiplex system to evaluate the efficacy of combined drug therapies aimed at preventing or delaying the emergence of resistant cells. Through a similar approach, we assessed the effects of repairing oncogenic driver mutations in addicted cancer cells directly at the genome level. Finally, we used a highly complex set of CRISPR-barcodes to trace intratumor heterogeneity, as a convenient alternative strategy to lentiviral barcode libraries.ConclusionCRISPR-barcoding is a fast and highly flexible means to investigate the effects of different kinds of genomic modifications in a broad range of functional assays.

Proliferating EpCAM-Positive Ductal Cells in the Inflamed Liver Give Rise to Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) originates from regenerating liver cells with genetic alterations in chronically inflamed liver. Ductal cells and hepatocytes proliferate for liver regeneration, and proliferating ductal cells (PDC) derived from bile ductules have long been considered putative liver stem/progenitor cells and candidate cellular origins of HCC. The potential of PDC as tumor-originating cells, however, remains controversial in contrast to accumulating evidence that HCC originates from hepatocytes. Here, we demonstrate that PDCs expressing the established surface and cancer stem cell marker EpCAM give rise to HCC in inflamed liver. EpCAM-expressing PDCs were specifically labeled in newly developed EpcamCreERT2 mice and traced in a chemically induced liver injury model. Stepwise accumulation of genetic alterations in EpCAM-positive cells was induced by the mutagenesis activity of activation-induced cytidine deaminase using conditional transgenic mice. Lineage-tracing experiments revealed that labeled PDC differentiated into cholangiocytes, but not into hepatocytes, in the chemically damaged liver. Nevertheless, EpCAM-positive PDC with genetic alterations gave rise to HCC after 8 months of chemical administration. PDC-derived HCC showed histologic characteristics of concomitant ductule-like structures resembling human cholangiolocellular carcinoma (CLC) and exhibited serial transitions from PDC-like CLC cells to hepatocyte-like HCC cells. The Wnt signaling pathway was specifically upregulated in the CLC components of PDC-derived HCC. Our findings provide direct experimental evidence that EpCAM-expressing PDC could be a cellular origin of HCC, suggesting the existence of stem/progenitor-derived hepatocarcinogenesis. Cancer Res; 77(22); 6131-43. ©2017 AACR.

Helicobacter pylori-Mediated Genetic Instability and Gastric Carcinogenesis.

Helicobacter pylori infection is the most important cause of human gastric cancer worldwide. Gastric cancer develops over a long time after H. pylori infection via stepwise accumulation of genetic alterations and positive selection of cells with growth advantages. H. pylori itself and the resultant chronic inflammation lead to the emergence of genetic alterations in gastric epithelial cells via increased susceptibility of these cells to DNA damage. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) in inflammatory and gastric epithelial cells, as well as the expression of cytidine deaminase in gastric epithelial cells, may link H. pylori-related inflammation and DNA damage. Recent comprehensive analyses of gastric cancer genomes provide clues for the possible molecular mechanisms of gastric carcinogenesis. In this chapter, we describe how genetic alterations emerge during gastric carcinogenesis related to H. pylori infection.

Genomic Loss of DUSP4 Contributes to the Progression of Intraepithelial Neoplasm of Pancreas to Invasive Carcinoma

The progression from precursor lesions of pancreatic cancer, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN), to invasive disease is characterized by stepwise accumulation of genetic alterations. However, it remains unclear whether additional alterations are required for the progression of high-grade neoplasms to invasive pancreatic carcinoma. We compared the genomic profiles of paired noninvasive and invasive carcinoma tissues collected from patients with IPMN. We demonstrate that the frequency of genomic copy-number aberrations significantly increased during the course of invasion, and the loss of 8p11.22-ter was more often associated with invasive tissues. Expression profiling in pancreatic cancer cell lines with and without 8p11.22-ter revealed that DUSP4, an MAPK phosphatase, was significantly downregulated in cells lacking 8p11.22-ter as well as in invasive carcinomas due to genomic loss. Restoration of DUSP4 expression in pancreatic cancer cells significantly suppressed invasiveness and anoikis resistance via ERK inactivation. Accordingly, we found that blockade of ERK signaling by MEK inhibition was effective in an orthotopic xenograft model and significantly extended survival. Collectively, our findings demonstrate a genetic mechanism by which pancreatic precursor lesions progress to invasive carcinomas and highlight DUSP4 as a novel invasion suppressor that can be therapeutically exploited through manipulation of ERK signaling. Cancer Res; 76(9); 2612-25. ©2016 AACR.

Tumor suppressor microRNAs: A novel non‐coding alliance against cancer

Tumor initiation and progression are the outcomes of a stepwise accumulation of genetic alterations. Among these, gene amplification and aberrant expression of oncogenic proteins, as well as deletion or inactivation of tumor suppressor genes, represent hallmark steps. Mounting evidence collected over the last few years has identified different populations of non-coding RNAs as major players in tumor suppression in almost all cancer types. Elucidating the diverse molecular mechanisms underlying the roles of non-coding RNAs in tumor progression might provide illuminating insights, potentially able to assist improved diagnosis, better staging and effective treatments of human cancers. Here we focus on several groups of tumor suppressor microRNAs, whose downregulation exerts a profound oncologic impact and might be harnessed for the benefit of cancer patients.

Keratinocyte Stem Cells and the Targets for Nonmelanoma Skin Cancer†

The mammalian skin is a complex dynamic organ composed of thin multilayered epidermis and a thick underlying connective tissue layer dermis. The epidermis undergoes continuous renewal throughout life. The stems cells uniquely express particular surface markers utilized for their identification, isolation and localization in specific niches in epidermis as well as hair follicles (HFs). The two stage skin carcinogenesis model involves stepwise accumulation of genetic alterations and ultimately leading to malignancy. Whereas early research on skin carcinogenesis focused on the molecular nature of carcinogens and tumor promoters, more recent studies have focused on the identification of the target cells and tumor promoting cells for both chemical and physical carcinogens and promoters. Recent studies support the hypothesis that keratinocyte stem cells are the targets in skin carcinogenesis. In this review, we discuss briefly the localization of stem cells in the epidermis and HFs, and review the possibility that skin papillomas and carcinomas are derived from stem cells, as well as from other cells in the cutaneous epithelium whose stem cell properties are not well known.

Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE

BackgroundCancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).ResultsThe stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot.ConclusionThese findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis.

REIC/Dkk-3 induces cell death in human malignant glioma

The progression of glioma to more malignant phenotypes results from the stepwise accumulation of genetic alterations and the consequent disruption of the apoptotic pathway and augmentation of survival signaling. REIC/Dkk-3, a member of the human Dickkopf (Dkk) family, plays a role as a suppressor of the growth of several human cancers; however, to date it has not been identified in brain tumors. We compared the gene and protein expression of REIC/Dkk-3 in human malignant glioma and normal brain tissues using quantitative real-time PCR, Western blotting, and immunohistochemistry. We also performed small interfering REIC/Dkk-3 (siREIC/Dkk-3) knockdown and REIC/Dkk-3 overexpression experiments to examine the role of REIC/Dkk-3 in human malignant glioma cells in vitro. In brain tissue from patients with malignant glioma, the gene and protein expression of REIC/Dkk-3 was lower than in normal brain tissue and was related to the malignancy grade. In the primary glioblastoma cell line, REIC/Dkk-3 transfection led to apoptosis owing to the activation of phosphorylated JUN, caspase-9, and caspase-3 and the reduction of beta-catenin; in REIC/Dkk-3 knockdown experiments, cell growth was augmented. Our results suggest that REIC/Dkk-3 regulates the growth and survival of these cells in a caspase-dependent and -independent way via modification of the Wnt signaling pathway. Our work is the first documentation that the gene and protein expression of REIC/Dkk-3 is down-regulated in human malignant glioma. Our demonstration of the mechanisms underlying REIC/Dkk-3-induced cell death indicates that REIC/Dkk-3 plays a pivotal role in the biology of human malignant glioma and suggests that REIC/Dkk-3 is a promising candidate for molecular target therapy.

Multilocus loss of heterozygosity allelotypes identify a genetic pathway associated with progression from low to high stage disease in neuroblastoma

Neuroblastoma is a heterogeneous tumour with a variety of clinical phenotypes, ranging from a localised tumour with excellent outcome (stage 1) to a metastatic, usually fatal malignancy (stage 4). In order to investigate the genetic relationship between these tumour subtypes, a loss of heterozygosity (LOH) analysis was carried out. Composite LOH allelotypes incorporating data from 96 loci on 5 chromosomes (1p, 3p, 4p, 11q, 14q), were constructed for 62 neuroblastomas. Neuroblastomas with similar allelotypes were clustered into groups and allelotype patterns correlated with clinical features. Three distinct genetic subgroups of neuroblastoma were observed. The largest group (50% of tumours) was characterised by specific allelotype patterns indicative of a stepwise accumulation of genetic alterations (11q LOH → 1p, 4p, and/or 14q LOH → 3p LOH), associated with progression from low to high stage disease. These tumours are distinct from MYCN amplified neuroblastomas which have a more rapid and aggressive disease course, and also a proportion of low stage tumours, often ganglioneuromas or ganglioneuroblastomas, with restricted growth potential.

Evolution of B-cell clonal expansions with age

B-cell clonal expansions (BCE) in young mice are transient, detectable for less than 4 weeks. In contrast, BCE in old mice persist more than 2 months. The greater persistence of BCE in old mice does not appear to be due to the age of the host as the survival of phenyloxazolone chicken serum albumin-induced BCE in most old mice was shorter than in young mice. This raises the possibility that persistent BCE seen in old mice develop over time from transient BCE present earlier in life. To test this hypothesis, young C57BL/6 mice were immunized with hen egg lysozyme (HEL) during the first year of life. By 28 months of age, the majority of these mice had developed a benign, persistent BCE associated with a HEL-specific serum mIg. We also investigated whether benign, persistent BCE, present in 18-month-old mice, can evolve into B-cell lymphomas. We observed that four of eight C57BL/6 mice that survived to 29 months of age had developed diffuse large cell lymphomas. In three of these mice, this diagnosis was made by microscopic analysis of the lymphoid organs. In one mouse, a macroscopic lymphoma was present that permitted us to demonstrate that the IgH mRNA CDR3 length and sequence in the malignant lymphoma was derived from a persistent BCE present 11 months earlier. Together these observations are consistent with the hypothesis that stepwise accumulation of genetic alterations combined with Darwinian selection underlies the evolution of B cells from transient BCE in young mice into persistent BCE, serum mIg, and B-cell lymphomas observed in older mice.

Chromosomal aberrations related to metastasis of human solid tumors.

The central role of sequential accumulation of genetic alterations during the development of cancer has been firmly established since the pioneering cytogenetic studies successfully defined recurrent chromosome changes in specific types of tumor. In the course of carcinogenesis, cells experience several genetic alterations that are associated with the transition from a preneoplastic lesion to an invasive tumor and finally to the metastatic state. Tumor progression is characterized by stepwise accumulation of genetic alterations. So does the dominant metastatic clone. Modern molecular genetic analyses have clarified that genomic changes accumulate during the development and progression of cancers. In comparison with the corresponding primary tumor, additional events of chromosomal aberrations (including gains or allelic losses) are frequently found in metastases, and the incidence of combined chromosomal alterations in the primary tumor, plus the occurrence of additional aberrations in the distant metastases, correlated significantly with decreased postmetastatic survival. The deletions at 3p, 4p, 6q, 8p, 10q, 11p, 11q, 12p, 13q, 16q, 17p, 18q, 21q, and 22q, as well as the over-representations at 1q, 8q, 9q, 14q and 15q, have been found to associate preferentially with the metastatic phenotype of human cancers. Among of them, the deletions on chromosomes 8p, 17p, 11p and 13p seem to be more significant, and more detail fine regions of them, including 8p11, 8p21-12, 8p22, 8p23, 17p13.3, 11p15.5, and 13q12-13 have been suggested harboring metastasis-suppressor genes. During the past decade, several human chromosomes have been functionally tested through the use of microcell-mediated chromosome transfer (MMCT), and metastasis-suppressor activities have been reported on chromosomes 1, 6, 7, 8, 10, 11, 12, 16, and 17. However, it is not actually known at what stage of the metastatic cascade these alterations have occurred. There is still controversial with the association between the chromosomal aberrations and the metastatic phenotype of cancer. As the progression of human genome project and the establishment of more and more new techniques, it is hopeful to make clear the genetic mechanisms involved in the tumor metastasis in a not very long future, and provide new clues to predicting and controlling the metastasis.

Accumulation of Genetic Alterations in Brain Metastases of Sporadic Breast Carcinomas Is Associated with Reduced Survival after Metastasis

Tumor progression is characterized by stepwise accumulation of genetic alterations. To identify alterations associated with breast cancer metastasis, an analysis of comparative loss of heterozygosity (LOH) was performed on 38 primary sporadic breast carcinomas and 16 distant metastases. Two loci at 5q21 and 18q21 were chosen because of their reported increased deletion frequency in metastatic tumors. LOH at 17q21, 13q12–13, 17p13.1 and 11q22–23 was analyzed to determine whether there is a specific involvement of these breast cancer-associated gene loci in the metastatic process. Our data show that distant metastases are characterized by markedly increased LOH frequency at all loci examined. In both gene locus groups, significantly more distant metastases are affected by combined LOH. Furthermore, a significantly reduced postmetastatic survival time has been observed in patients with brain metastases affected by synchronous allelic loss at the four breast cancer-associated gene loci. Our results suggest that cumulative LOH of breast cancer-related gene loci is associated with a more aggressive phenotype of metastatic breast tumors.