CRISPR Cas9 in Pancreatic Cancer Research

Pancreatic cancer is now becoming a common cause of cancer death with no significant change in patient survival over the last 10 years. The main treatment options for pancreatic cancer patients are surgery, radiation therapy and chemotherapy, but there is now considerable effort to develop new and effective treatments. In recent years, CRISPR/Cas9 technology has emerged as a powerful gene editing tool with promise, not only as an important research methodology, but also as a new and effective method for targeted therapy. In this review, we summarize current advances in CRISPR/Cas9 technology and its application to pancreatic cancer research, and importantly as a means of selectively targeting key drivers of pancreatic cancer

Cancer Is Associated with Alterations in the Three-Dimensional Organization of the Genome

The human genome is organized into topologically associating domains (TADs), which represent contiguous regions with a higher frequency of intra-interactions as opposed to inter-interactions. TADs contribute to gene expression regulation by restricting the interactions between their regulatory elements, and TAD disruption has been associated with cancer. Here, we provide a proof of principle that mutations within TADs can be used to predict the survival of cancer patients. Specifically, we constructed a set of 1467 consensus TADs representing the three-dimensional organization of the human genome and used Cox regression analysis to identify a total of 35 prognostic TADs in different cancer types. Interestingly, only 46% of the 35 prognostic TADs comprised genes with known clinical relevance. Moreover, in the vast majority of such cases, the prognostic value of the TAD was not directly related to the presence/absence of mutations in the gene(s), emphasizing the importance of regulatory mutations. In addition, we found that 34% of the prognostic TADs show strong structural perturbations in the cancer genome, consistent with the widespread, global epigenetic dysregulation often observed in cancer patients. In summary, this study elucidates the mechanisms through which non-coding variants may influence cancer progression and opens new avenues for personalized medicine

Chemoresistance in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC), generally known as pancreatic cancer (PC), ranks the fourth leading cause of cancer-related deaths in the western world. While the incidence of pancreatic cancer is displaying a rising tendency every year, the mortality rate has not decreased significantly because of late diagnosis, early metastasis, and limited reaction to chemotherapy or radiotherapy. Adjuvant chemotherapy after surgical resection is typically the preferred option to treat early pancreatic cancer. Although 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel can profoundly improve the prognosis of advanced pancreatic cancer, the development of chemoresistance still leads to poor clinical outcomes. Chemoresistance is multifactorial as a result of the interaction among pancreatic cancer cells, cancer stem cells, and the tumor microenvironment. Nevertheless, more pancreatic cancer patients will benefit from precision treatment and targeted drugs. Therefore, we outline new perspectives for enhancing the efficacy of gemcitabine after reviewing the related factors of gemcitabine metabolism, mechanism of action, and chemoresistance

Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Abstract Background Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic "control mechanisms". Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes. Methods From literature search we identified the anti-apoptotic genes XIAP, Survivin and Bcl-2 as commonly upregulated in pancreatic cancer. We performed SGS and silencing of single candidate genes using siRNA molecules in two pancreatic cancer cell lines. Effectiveness of SGS was assessed by qRT-PCR and western blotting. Apoptosis induction was measured by flow cytometry and caspase activation. Results Simultaneous gene silencing reduced expression of the three target genes effectively. Compared to silencing of a single target or control, SGS of these genes resulted in a significant higher induction of apoptosis in pancreatic cancer cells. Conclusions In the present study we performed a subliminal silencing of different anti-apoptotic target genes simultaneously. Compared to silencing of single target genes, SGS had a significant higher impact on apoptosis induction in pancreatic cancer cells. Thereby, we give further evidence for the concept of an anti-apoptotic buffering capacity of pancreatic cancer cells.</p

Silenced ZNF154 Is Associated with Longer Survival in Resectable Pancreatic Cancer

Pancreatic cancer has become the third leading cause of cancer-related death in the Western world despite advances in therapy of other cancerous lesions. Late diagnosis due to a lack of symptoms during early disease allows metastatic spread of the tumor. Most patients are considered incurable because of metastasized disease. On a cellular level, pancreatic cancer proves to be rather resistant to chemotherapy. Hence, early detection and new therapeutic targets might improve outcomes. The detection of DNA promoter hypermethylation has been described as a method to identify putative genes of interest in cancer entities. These genes might serve as either biomarkers or might lead to a better understanding of the molecular mechanisms involved. We checked tumor specimens from 80 patients who had undergone pancreatic resection for promoter hypermethylation of the zinc finger protein ZNF154. Then, we further characterized the effects of ZNF154 on cell viability and gene expression by in vitro experiments. We found a significant association between ZNF154 hypermethylation and better survival in patients with resectable pancreatic cancer. Moreover, we suspect that the cell growth suppressor SLFN5 might be linked to a silenced ZNF154 in pancreatic cancer

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells

A genome-wide map of aberrantly expressed chromosomal islands in colorectal cancer

BACKGROUND: Cancer development is accompanied by genetic phenomena like deletion and amplification of chromosome parts or alterations of chromatin structure. It is expected that these mechanisms have a strong effect on regional gene expression. RESULTS: We investigated genome-wide gene expression in colorectal carcinoma (CRC) and normal epithelial tissues from 25 patients using oligonucleotide arrays. This allowed us to identify 81 distinct chromosomal islands with aberrant gene expression. Of these, 38 islands show a gain in expression and 43 a loss of expression. In total, 7.892 genes (25.3% of all human genes) are located in aberrantly expressed islands. Many chromosomal regions that are linked to hereditary colorectal cancer show deregulated expression. Also, many known tumor genes localize to chromosomal islands of misregulated expression in CRC. CONCLUSION: An extensive comparison with published CGH data suggests that chromosomal regions known for frequent deletions in colon cancer tend to show reduced expression. In contrast, regions that are often amplified in colorectal tumors exhibit heterogeneous expression patterns: even show a decrease of mRNA expression. Because for several islands of deregulated expression chromosomal aberrations have never been observed, we speculate that additional mechanisms (like abnormal states of regional chromatin) also have a substantial impact on the formation of co-expression islands in colorectal carcinoma

Application of microfluidic chips in anticancer drug screening

With the continuous development of drug screening technology, new screening methodologies and technologies are constantly emerging, driving drug screening into rapid, efficient and high-throughput development. Microfluidics is a rising star in the development of innovative approaches in drug discovery. In this article, we summarize the recent years' progress of microfluidic chip technology in drug screening, including the developmental history, structural design, and applications in different aspects of microfluidic chips on drug screening. Herein, the existing microfluidic chip screening platforms are summarized from four aspects: chip structure design, sample injection and drive system, cell culture technology on a chip, and efficient remote detection technology. Furthermore, this review discusses the application and developmental prospects of using microfluidic chips in drug screening, particularly in screening natural product anticancer drugs based on chemical properties, pharmacological effects, and drug cytotoxicity.Peer reviewe