MUC13 Enhances Colorectal Cancer Metastasis

Colorectal cancer (CRC) is one of the most prevalent cancer worldwide with a 5% lifetime incidence in developed countries. It is third most common cause of cancer related death in the United States and the second deadliest when men and women are combined. Encouragingly due to changes in dietary lifestyle, screening colonoscopy, and advancement in treatments the mortality has decreased in recent years. Most sporadic CRCs develop from polyploid adenomas and are preceded by intramucosal carcinomas (stage 0), which can progress into more malignant forms. This developmental process is known as the adenoma-carcinoma sequence. Early detection and endoscopic removal are crucial for CRC management. The overall 5-year survival of CRC jumps noticeably from 66% to 91% if it can be diagnosed and treated at early stages but drops down to 14% when the disease has metastasized. Metastasis is the cause of 90% of all cancer related deaths, which necessitates the need to understand the mechanisms regulating different progression steps. Metastasis is a complex and multistep process, whereby cancer cells leave the primary tumor and colonize in new tissues. However, only about 0.02% of tumor cells that detach from the primary tumor are successful in forming a metastatic lesion. After intravasation, anchorage independent survival (Anoikis resistance) of primary tumor cell is one of the key steps in metastasis. The mucin MUC13 when aberrantly overexpressed in cancer has been found to lead to poor outcomes in Pancreatic, Ovarian, Liver, and Colorectal cancer. When overexpressed in these cancers MUC13 has been shown to increase the oncogenic activity and survival of cancer cells. The oncogenic nature of MUC13 indicates a possible role in the development of Anoikis resistance and CRC metastasis. To understand the mechanism behind Anoikis resistance we developed and optimized an Anoikis induction model using the low adhesion poly hema coated cell culture dishes. Two isogenic cell lines SW480 (primary tumor site) and SW620 (metastatic tumor site) were used for this study. In our initial experiments SW620 demonstrated less cell death compared to SW480 during Anoikis induction. High MUC13 expression was observed in SW620 between 24-36 hours compared at 0 hours. SW620 further showed an increase in the expression of anti-apoptotic protein Bcl2 also. Overexpression of MUC13 in SW480 (SW480+MUC13) cells increased oncogenic phenotype such as invasion, migration, proliferation, and colony size as compared to SW480+Vector control cells. Downregulation of MUC13 in SW620 cells resulted in decreased oncogenic traits. The role of MUC13 in Anoikis resistance was further investigated in the overexpression and knockdown cell lines. When overexpressing MUC13 in SW480 cells we found increased survival compared to SW480+Vector cells. Bcl2 expression increased while Cleaved-Caspase 3 showed decreased expression in SW480+MUC13 compared to SW480+Vector cells. Increased MUC13 expression also increased the metastatic potential of SW480 cells in mice. After 36 hours of Anoikis induction and then injection into mice through the tail vein, MUC13 mice had an increased tumor burden compared to Vector mice, with METs forming in the liver, kidneys, and lungs. This would indicate that MUC13 plays a larger part in CRC metastasis and specific in the development of Anoikis resistance. To understand the mechanism involving MUC13 in Anoikis resistance, we studied kinases after Anoikis induction in the cell lines, followed by quantitative proteomics. We were able to find changes in transcription factor YAP1 and YAP1 and β-catenin, which are known to play vital roles in cellular development. In recent studies YAP1 and β-catenin have been shown to form a complex that promotes tumor cell survival and increased tumorigenesis. High MUC13 expression between 24-36hrs was observed, leading to an increase in nuclear localization of the known survival complex YAP1/β-catenin. Further in-vitro analysis indicated that MUC13 plays a vital role through direct cooperation with YAP1 and β-catenin, and together with increased nuclear localization of YAP1/MUC13 and β-catenin/MUC13 complexes, results in increase expression of pro-survival genes. This relationship was further examined and validated in an in-vivo mouse model, in which high MUC13 expression led to increased YAP1 and β-catenin expression and tumorigenesis. A correlation was further observed between MUC13 and YAP1 expression in human CRC patient tissue samples with high expression in tumor tissues compared to NAT and increased nuclear localization with elevated expression for both MUC13 and YAP1. The novel interaction between YAP1 and MUC13 defines a new mechanism in which cells develop Anoikis resistance in CRC. Genetic variations in genes are a well-known aspect of most diseases. This is especially true for cancer. Genetic variations in mucins such as MUC1 and MUC5AC have been found to increase the risk of stomach cancer and certain Allele mutations nearly doubling that risk in. We investigated if MUC13 had any genetic variants and found 5 different splice variants. Three of those splice variants were found to be non-coding. We then investigated the two protein coding variants consisted of a 512aa (Long form) and 187aa (Short form). We found that MUC13-LF was responsible for generating the oncogenic phenotype associated with adherent MUC13 expression. MUC13-SF however, showed a decrease in migration and invasion when overexpressed in MUC13 null cell lines. These studies suggest an oncogenic function of MUC13 in CRC via influencing multiple signaling pathways however, its role in cancer metastasis is remains elusive. In this study, we discovered how MUC13 facilitates metastasis after dissemination of tumor cells from the primary tumor site through influencing interaction and nuclear translocation of YAP1 and β-catenin followed by the expression of their downstream pro-survival and metastasis genes. After escaping from the primary tumor this MUC13 driven molecular mechanism provides a crucial survival advantage to anchorage independent circulating tumor cells, leading to successful and extravasation and homing new distant site for cancer metastasis. The interaction between YAP1 and MUC13 provides a new therapeutic in the prevention of metastasis that in combination with current chemotherapy could limit the tumor to its primary site

Recent advances on the role of long non-coding RNAs in Alzheimer’s disease

Dementia is a progressive cognitive impairment that affects the activities of daily living. Alzheimer’s disease (AD) is the most common form of the dementia worldwide accounting for 60–80% of all dementia cases. With an estimated cost exceeding $290 billion in the USA, understanding and development of future therapeutic strategies is vital. In this perspective, we will be examining the current thinking of AD research and therapeutic strategies, while proposing a possible new direction for diagnosis, understanding, and treatment targets. Non-coding RNA accounts for the largest population of the human transcriptome. Long noncoding RNA (lncRNA) is a recent molecule of interest in the biomedical research which is non protein coding and is of length greater than 200 nucleotides. LncRNAs have been shown to play diverse roles within the cells such as posttranscriptional and posttranslational regulation, chromatin modulation, and protein complex organization. Given the flexible and diverse role in disease pathophysiology, lncRNAs may serve as novel therapeutic targets for diagnosis and treatment. Evidently, recent studies showed that dysregulation of lncRNA influences the clinical course of tumorigenesis, neurological disorders, cardiovascular disease, diabetes, and acquired immunodeficiency syndrome (Kazimierczyk et al., 2020). This indicates that lncRNA can provide a unique avenue of research and possible therapeutic targets in AD

Biophysical Changes Caused by Altered MUC13 Expression in Pancreatic Cancer Cells

Background Pancreatic cancer is one of the most lethal cancers in the United States. This is partly due to the difficulty in early detection of this disease as well as poor therapeutic responses to currently available regimens. Our previous reports suggest that mucin 13 (MUC13, a transmembrane mucin common to gastrointestinal cells) is aberrantly expressed in this disease state, and has been implicated with a worsened prognosis and an enhanced metastatic potential in PanCa. However, there is virtually no information currently to describe the biophysical ramifications of this protein. Methods To demonstrate the biophysical effect of MUC13 in PanCa, we generated overexpressing and knockdown model cell lines for PanCa and subsequently subjected them to various biophysical experiments using atomic force microscopy (AFM) and cellular aggregation studies. Results AFM-based nanoindentation data showed significant biophysical effects with MUC13 modulation in on PanCa cells. The overexpression of MUC13 in Panc-1 cells led to an expected decrease in modulus, and a corresponding decrease in adhesion. With MUC13 knockdown, HPAF-II cells exhibited an increased modulus and adhesion. These results were confirmed with altered cell-cell adhesion as seen with aggregation assays. Conclusions MUC13 led to significant biophysical changes in PanCa cells and which exhibited characteristic phenotypic changes in cells demonstrated in previous work from our lab. This work gives insight into the use of biophysical measurements that could be used to help diagnose or monitor cancers as well as determine the effects of genetic alterations at a mechanical level

Plasmid Midiprep: A Method to Purify Plasmids for Recombinant DNA Studies

A fundamental aspect of molecular biology involves exploring the properties and functions of specific genes. The rise of recombinant DNA technology has vastly improved and simplified functional studies by allowing scientists to isolate specific genes using restriction enzymes and plasmids providing greater precision. Plasmids take great significance in downstream studies, which is why quantity and quality of the plasmids purified is important. In this study, we isolated and purified recombinant plasmids in microgram quantities to confirm the yield, quantity, and quality using spectral and size fractionation methods. We also assessed the plasmids for application in downstream studies. We found the plasmids that were isolated with a good yield ranging from 1-1.5 mg/ml. The high yield and purity suggest the Promega Midiprep kit is effective in producing high quality plasmids. The size of the plasmids was assessed using gel electrophoresis, and a transient transfection into mammalian cells confirmed their expression through fluorescence

Long non-coding RNA (lncRNA) as a new biomarker for hepatocellular carcinoma (HCC) drug resistance

Background: Hepatocellular carcinoma (HCC) is the 4th leading cause of cancer-related deaths worldwide and the 6th most common cancer worldwide. When HCC progresses to advanced stages, drug resistance becomes a major hurdle and leaves clinicians with limited therapeutic options. Long non-coding RNAs (lncRNAs) have shown to promote drug resistance in various cancers. The goal of our research is to explain the molecular role of lncRNAs in HCC drug resistance and compile a comprehensive list of studied lncRNAs involved in HCC drug resistance. Methods: To compile a list of lncRNA involved in HCC drug resistance we performed an advanced search on Lnc2Cancer, a database that provides experimentally supported associations between lncRNA and human cancer, using the following filters: “hepatocellular carcinoma”, “drug clinical application”, “lncRNA”, “all biological function”, and “all regulatory mechanism.” Results: We identified 12 lncRNAs that are involved in HCC drug resistance: Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT 1), Keap1 Regulation-Associated LncRNA (KRAL), Transcribed Ultra-conserved Region 338 (TUC338), Long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR), Linc-VLDLR, Highly Upregulated in Liver Cancer (HULC), HCC associated long non-coding RNA (HANR), LncRNA Regulator of AKT Signaling Associated with HCC and RCC (LncARSR), Taurine up-regulated gene 1 (TUG1), H19, NR2F1 Antisense RNA 1 (NR2F1-AS1), and HOX Transcript Antisense RNA (HOTAIR). Conclusions: Our review demonstrates that lncRNAs involved in HCC drug resistance participate in various mechanistic categories such as autophagy, epithelial-mesenchymal transition, and efflux pump upregulation. There is a need to uncover novel lncRNA biomarkers for both the early detection of HCC and to create drug strategies for clinicians when predicting chemoresistance

Characterization of POTE-2 Expression in Hepatocellular Carcinoma Cell Lines

Hepatocellular carcinoma is the most common cancer of the liver and is the third leading cause of cancer deaths globally. Due to the limited serum biomarkers, detection of hepatocellular carcinoma usually occurs in the later, metastasized stages of the disease where the 5-year survival rate falls to around 3%. The identification of early diagnostic biomarkers for hepatocellular carcinoma is necessary to provide both improved prognostic outcomes and make treatment options dependent on liver dysfunction, such as resection and liver transplantation, available to patients. This study performed real-time polymerase chain reaction and western blot analysis for four hepatocellular carcinoma cell lines (SKHEP1, HEP3B, C3A, and HEPG2) to observe the expression of POTE-2 mRNA and protein. The data demonstrates significant overexpression of POTE-2 in HCC cell lines, indicating these HCC cell lines to be an ideal in-vitro model to study POTE-2 function in hepatocellular carcinoma

Performing a High-Throughput Virtual Screening (HVTS) to identify potential therapeutic targets of YB-1 protein

Background: Hepatocellular carcinomas (HCCs) is a primary malignancy of the liver. Hispanic-Texans have several risk factors and disparities that compound the risk of HCC diagnosis and treatment. The most used chemotherapeutic drug against HCC is sorafenib, but many liver cancers have developed a resistance to this drug. The knockdown of Y-box binding protein-1 (YB-1) has been shown to greatly increase sensitivity to sorafenib. In this study, we will discuss identification of potential YB-1 inhibitors, which can lead to re-sensitization of liver cancer cells to sorafenib. Methodology: The RCSB protein data bank (pdb) was used to retrieve the crystal structure of YB1, while the DrugBank database was used to obtain a list of experimental and approved drugs. A multiple sequence alignment (MSA) of YB-1 & Lin28 was done by Clustal Omega. Biovia Discovery Studio 2020 was used to visualize 3D models and perform a High-Throughput Virtual Screening (HTVS), which includes rigid docking via the LibDock extension, flexible docking via the CDocker extension, and a pharmacokinetic profiling via an ADMET analysis. Results: The cold shock domain of YB-1 was found to be conserved with Lin28, as a known transcription factor. 22 drug candidates were identified through HTVS. The best six show a decent binding ability in both rigid and flexible dockings and have been previously tested in different cancer types to some extent. Conclusion: We were able to identify six potential drug candidates for inhibiting our protein of interest, YB-1. Studies are in progress to study them on sorafenib-resistant HCC cell lines

Mucin MUC13 and YAP1 correlate with poor survival in colorectal cancer

Background: Metastatic disease contributes to over 90% of cancer-associated deaths. Colorectal cancer (CRC), the second lethal malignancy, has the greatest incidence and mortality rates in the Southern United States. Over 40-50% of CRC patients acquire metastasis at some point throughout their disease\u27s progression. CRC survival rate drops from 90%-14% when the disease is confined within the colon and therefore “early diagnosis” becomes imperative to determine timely and quality treatments. We have identified that MUC13 protein translocate to nucleus along with transcription factor Yes-Associated Protein 1 (YAP1) during anchorage independent conditions (metastatic phenotype). YAP1 is known to be overexpressed in CRC which promotes proliferation and survival of CRC cells. This study will provide information regarding MUC13 and YAP1 correlation and their role in CRC patient outcomes. Methods: The comparative analysis of MUC13 and YAP1 expression in CRC samples (Tissue Microarrays (TMA) of CRC patients (39 cases and 95 cores)) with Pathology grade, TNM Classification, Clinical stage, and Survival information were investigated using Immunohistochemistry (IHC) staining, followed by digital scanning by 3D-Histech scanner, and analysis using QuantCenter image analysis software. Results: IHC analysis revealed increased MUC13 expression in colon adenocarcinoma and metastatic adenocarcinoma compared to normal colon tissues. MUC13 expression was observed in nucleus, cytoplasm and membrane associated with mostly with poorly differentiated adenocarcinomas, while YAP1 was localized in the nucleus. The correlation of MUC13/YAP1 expression with patient outcome is in progress. Conclusion: This study will potentially establish a correlation between MUC13 and YAP1 with CRC patient outcome

Role of POTE-2 in hepatocellular carcinoma progression.

Background: Hepatocellular carcinoma (HCC) accounts for 85-90% of primary liver cancers. The Hispanic population had an incidence of 21.2 per 100,000 in Texas. Particularly, the Rio Grande Valley (RGV) is an underserved area facing disparities that increase risk factors of HCC and thus, yielding higher incidence and mortality. Therefore, early, faster, and inexpensive diagnostic biomarkers and methods are crucial to under-resourced areas such as the RGV. Recently, we have identified an extracellular cancer antigen, POTE-2. Preliminary data indicates high POTE-2 expression in HCC tumors. In this study, we will discuss the role of POTE-2 in HCC progression and its associated regulatory pathways. Methods: The Cancer Genome Atlas (TCGA) database of HCC patients (n=371 tumor; n=50 normal) was analyzed. Liver cancer cells were procured from ATCC. POTE-2 mRNA and protein expression analyzed via RT-PCR and western blot. Absolute copy number was determined using Digital Droplet PCR. Lentiviral-based plasmids were used for overexpression and knockdown studies. Signaling pathways were analyzed using Proteome Profiler array. Results: Comprehensive analysis of TCGA database revealed high POTE-2 expression tumors with upregulation in all stages of HCC. POTE-2 expression increases with nodal metastatic status leading to poor survival. The protein expression for POTE-2 was significantly higher in SK-HEP1 compared to C3A cells. Lentiviral transduction showed significant overexpression and knockdown of the POTE-2 protein. Modulation of POTE-2 expression led to changes in lncRNA and kinase pathways. Conclusion: These studies will help discover novel mechanisms of POTE-2 protein function, signaling pathways and roles in liver cancer progression

Anticancer drug screening using invitro Cell Proliferation assay

Introduction: In this presentation cell proliferation methods and how they are related to screening for effective chemotherapy drugs will be reviewed. Cancer in its most basic form is the unchecked mass dividing of cells while normal apoptosis is not undertaken for various reasons, some of which that have yet to be discovered. By these means’ tumors form that inhibit the functions of the organs it is residing in and the effected cells may metastasize and spread throughout the body. For this reason, chemotherapy drugs must be assessed through introduction into working strains of cultured cancer cells that are then screened for effectiveness through a process called cell proliferation assays. Objective: The goal is to find the exact dosage for inhibiting the greatest number of Colorectal Cancer Cell (CRC) cells, strain HCT-116, while leaving other healthy cells unaffected as the methods are explained. Methods: Several methods exist for determining the resulting levels of proliferation of cells after drug administration such as: Molecular Targeted Therapies (MTT) and (Water Soluble Tetrazolium) WST-1 that uses a tetrazolium salt reagent on the cells before introducing the drug in question and then a colorimetric assay is used to determine the quantity of living cells remaining through assessing which retain the dye that is produced. Alamar BLUE is another experiment that uses redox reactions but substitutes the tetrazolium with resorufin, other options include Bromodeoxyuridine (BrdU) assay which analyzes the amount of a thymidine analog that is present post experiment after it has been absorbed by denatured DNA. Results: Once cell counts have been gathered, drugs of varying concentrations have been administered, and the assays are performed to gather the number of cells that continue to proliferate, tables and graphs reflecting such information may be drawn to find the Growth Inhibitory dose of 50% (GI 50) of the cells. Discussion: At the conclusion of these trials work will still need to be done to find how these drugs will be implemented in vivo. The next logical step is moving on to testing these therapies on animals to find strengths and weaknesses in live models. Conclusion: Through the course of testing 4 different chemotherapy drugs on HCT-116, MTT and other such cell proliferation assays are utilized in finding the correct dosages to elicit the desired response of inhibiting the growth of CRC cells. The process will begin from the first splitting of cell stock to acquire workable amounts of HCT-116, culturing this working stock, and passaging it as the quantity of cells become larger. The cell proliferation methods will be outlined along with the reasoning and theory behind them to include the materials utilized. The results will be discussed while also explaining how the results are to be properly evaluated. Finally, graphed analysis resulting in either GI 50 curves may be constructed to better tabulate what the varying concentrations effects resulted in. From here we continue to narrow our search to more finite concentrations that will yield better results in killing only the exact number of cells desired