Presentation Previews / 'Read More About It' Pages - Biology 376 - Fall 2021

H-Ras oncogene counteracts the growth-inhibitory effect of genistein in T24 bladder carcinoma cells

The oncoprotein Ras, encoded by a member of the ras proto-oncogene family, where Ras transduces signals in the signaling pathway incorporating the route via raf-1, MEK, ERK, and inducing the expression of the early gene c-fos which subsequently turns on a cascade of downstream genes crucial to the cellular signaling processes. Provided that Ras is considered to be one of the most important targets to suppress cancer tumor cell growth, it is vital to note the oncogenic activation of Hras by which remains as a factor of the mutational effect that oncogenic Hras has on human T24 TCC (transitional cell carcinoma) cells of bladder cancer tumors. Human bladder tumor cell lines have been previously treated with genistein, a compound in soybean products that has the capability to suppress angiogenesis or inhibit tumor cell growth in vitro and in vivo, which remains as the focus of research due to the presence in clinical detection of this membrane protein in angiogenesis and associated bladder tumors. Bladder TCC cells readily undergo apoptosis induced by genistein through the p21WAF1/CIP1 expression-involved EGFR signaling pathway.

Previous studies have noted the impact of both TGFR upregulation in the expression of tumor cells of T24 human lines and the reversal of cancerous phenotypes by genistein in the transformation of cells by H-ras or Hras val 12 (given that mutations occur at codon 12). The anticancer effects of genistein have led researchers in the direction of how patients with urinary bladder neuroplasms can utilize this drug therapy given the gene expression alterations to human bladder tumor cells by the protein and mechanisms of growth inhibition meditated by the drug. The varying patterns of gene expression in these human cell lines, with both sensitive and resistant phenotypes to the drug treatment of genistein, were examined utilizing cDNA microarrays in order to determine the key genes of the cell growth regulatory pathway; and both RT-PCR (for envisioning gene expression of egr-1 and c-fos) and immunoblotting analyses (for revealing the targeted proteins from detections of the antibody-antigen complexes in the mix of antibodies against human H-Ras, c-fos, and egr-1) were used.

Upon cell treatment with anti-Hras ODN and dose-dependent genistein, the cell growth of the T24 cells indicated a further decreased level comparable to the rates of the TCC genistein-sensitive lines, which was further evidenced by the antisense experiment where antisense ODN inhibited cell growth to a point where overexpression of Hras val 12 was required in order to avoid susceptibility to genistein mediated-growth inhibition. The results demonstrate that of the 8 human bladder cell lines tested for susceptibility of genistein induced inhibition of tumor cell proliferation, solely the activated T24 cells harboring Hras were consistently resistant to the drug treatment; a finding that led to the conclusion that the signal transduction pathways induced by H-ras val12 are responsible for the resistant phenotype of T24 bladder TCC cells to the anticancer therapeutic drug.

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Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells

N-linked glycosylation is an important protein modification that can occur during or after translation. N-linked glycosylation is the addition of an oligosaccharide to the nitrogen of the amino acid Asparagine. Oligosaccharides are carbohydrates consisting of many sugar molecules - also known as glycans. This process is often seen with secreted proteins and will occur within and outside of the endoplasmic reticulum (ER). Relevant proteins that depend on N-linked glycosylation for normal functioning include receptor tyrosine kinase (RTK) proteins such as the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). RTKs are greatly studied in oncology as they are often oncogenes and are found in many different types of cancers. This study aimed to gain a better understanding between N-linked glycosylation and the function of RTKs. Simultaneously, it aimed to identify a new possible method of targeted RTK inhibition.

Tunicamycin is a known and natural substance that blocks N-linked glycosylation by inhibiting associated enzymes. However, it also leads to total cell death. Several enzymes are involved with this process of adding glycans to proteins, but of particular interest is the enzyme oligosaccharyltransferase (OTS). Researchers were interested in identifying other small molecules that also block N-linked glycosylation. To do this, they initiated a high-throughput screen (HTS) which yielded the cell-permeable molecule NGI-1.

Four conclusions were drawn about NGI-1 from this study. First, NGI-1 disrupts OTS function. Using western blots, researchers confirmed that NGI-1 targets the subunits of OTS and prevents some, but not all N-linked glycosylation from occurring. Second, NGI-1 blocks glycosylation of EGFR and its transport to the cell surface. Using western blots along with confocal microscopy and immunofluorescence of EGFR, researchers confirmed that NGI-1 blocked the transfer of some, but not all glycans to EGFR. Furthermore, the addition of NGI-1 resulted in the location of EGFR to be in the intracellular compartment opposed to the normal extracellular plasma membrane location. NGI-1 was found to be protein specific, as other observed proteins remained unaffected by its presence and migrated to the cell surface as normal. Third, NGI-1 blocks the proliferation of non-small-cell lung cancer (NSCLC) cells that are dependent on the RTK pathway. Using western blots and phosphoprotein arrays, researchers confirmed that NGI-1 prevents phosphorylation of mutated EGFR that is oncogenically signaling. In other words, it was found that NGI-1 has strong inhibitory effects on cell types in which proliferative signaling is driven by RTK proteins. Lastly, NGI-1 induces senescence in RTK- dependent lung cancer. With the addition of NGI-1 to NSCLC cells, evidence of senescence was observed. Evidence included an observed increase of the number of cells in the G1 phase of the cell cycle, reduction in apoptosis, reduction in cyclin D1 - a protein involved in initiating cell replication phase, and an increase in p21 - a protein that regulates the cell cycle.

These findings suggest that NGI-1 could possibly be used as an effective therapy for cancers in mammalian cells. Its reduced cell toxicity compared to tunicamycin suggests that it could be a safer and less invasive treatment.

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Axitinib blocks Wnt/β-catenin signaling and directs asymmetric cell division in cancer

Many cancers develop due to mutations in the Wnt/β-catenin signaling pathway. Of these cancers, most are due to mutations specifically in the genes for β-catenin and the APC destruction complex. There is currently no clinical use of anti-cancer therapies that target the Wnt pathways. This is due to the difficulty in distinguishing between Wnt cancer cells and adult stem cells (ASC). ASCs usually divide asymmetrically in order to produce separate daughter cells, while tumor precursor cells and malignant tumor cells divide symmetrically. There is new evidence that suggests these cancer cells aren't as limited to symmetrical division as we thought.

The goal of the experiment was to understand how the axitinib drug works along the Wnt/β-catenin pathway. Axitinib was able to inhibit Wnt signaling in cancer cells, even in the presence of mutations shared by cancer patients. It also mediates asymmetric cell division in cancerous cells, converting them from cells that are able to form tumors to a phenotype similar to ASCs. The authors called this phenomenon “Wnt ACD” (asymmetric cell division) and further tested its effects on prostate cancer cells.

Axitinib also degrades β-catenin independently of the APC destruction complex. This is the mechanism by which axitinib directs ACD: lower levels of β-catenin induced wnt-mediated ACD.

The authors had to identify the proteins that interact with axitinib, namely proteins in the nucleus that have degredation-associated domains. Previous literature suggested the importance of SHPRH as a DNA-repair protein. Through western blot analysis, the authors found axitinib to have a stabilizing effect on SHPRH at low concentrations. It was then concluded that SHPRH was necessary for axitinib to degrade B-catenin. Therefore, SHPRA negatively regulates the Wnt signaling pathway; overexpression of SHPRH led to a downregulation of Wnt, and therefore promoted ACD.

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Dec. 3 Presentations

ADMA mediates gastric cancer cell migration and invasion via Wnt/β-catenin signaling pathway

Main Goals: To explore the role of ADMA in gastric cancer. To determine if ADMA promotes GC migration and invasion via Wnt signaling pathway, they performed a series of experiments with different concentrations of ADMA support their hypothesis. To determine the relationship between ADMA and EMT in GC expansion. This will provide insight into the mechanism of ADMA in promoting GC invasion and metastasis. ADMA levels are high in patients with lung, gastric, breast, and esophageal cancer. Some studies suggest that ADMA can reduce cytotoxicity, reduce apoptosis and Caspase-3 activation and reverse the down regulation of bcl-2 expression. ADMA is known to play a significant role in promoting tumor progression in colon and esophageal cancer but its role in gastric cancer has not been well investigated

Materials and Methods: The specimens of 115 gastric cancer patients were analyzed by ELISA and survival analysis. Functional assays were used to assess the efects of ADMA on gastric cancer cells. Experiments were conducted to detect the signaling pathway induced by ADMA in GC.

Results: Gastric cancer patients with high ADMA levels had poor prognosis and low survival rate. Furthermore, high level of ADMA did not afect the proliferation but promoted the migration and invasion of gastric cancer cell. Moreover, ADMA enhanced the epithelial–mesenchymal transition (EMT). EMT enables cancer cells to acquire mobility, chemo-resistance, and the capacity to migrate from the primary site Importantly, ADMA positively regulated β-catenin expression in GC and promoted GC migration and invasion via Wnt/β-catenin pathway. EMT is modulated by intracellular cues and triggered by overexpressed transcription factors. These transcription factors that are downstream of many signaling pathways, including Wnt/B-catenin

Conclusions:ADMA regulates gastric cancer cell migration and invasion via Wnt/β-catenin signaling pathway which promotes EMT. The results of these experiments may be applied to clinical practice as a biomarker and targeted therapy.

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P53 Pathway is involved in cell competition during mouse embryogenesis

The p53 gene has been under continual intense investigation and has been deemed as the guardian of the genome. This Tp53 gene as we have already learned in class, is a gene that provides instructions for acting as a tumor suppressor by regulating cell division and keeping cells from proliferating too fast or uncontrollably. Regulation occurs by triggering cell cycle arrest, apoptosis and senescence; ultimately deciding fate of a cell on whether it lives or dies. P53 is closely regulated, it has been determined that posttranslational regulatory mechanisms are crucial for regulating the activation of this gene. Acute stressful conditions, such as DNA damage, create high levels of p53 activity. However, p53 activity on low level stress has been mainly unexplored. This paper focuses on studying this specific literature gap.

The researchers focused their study on two very important negative regulators of p53 which are murine double minute regulators (MDM2 and MDM4) which maintain low activity of p53 in physiological conditions. MDM2 acts as an E3 ubiquitin ligase which its main function is to degrade p53. MDM4 functions as a homolog and inhibits the gene of interest by making a transcriptional activation domain.

Researchers used haploinsufficiency of MDM2 and MDM4 to induce mild p53 activation in vivo. They ultimately discovered that double heterozygous mice presented with growth disadvantages and were outcompeted by wild-type mice. More importantly, they discovered that out-competition in mice was dependent on the level of p53 activity. Cells with mild p53 activation resulted in lesser fit phenotypes. Researchers can to the conclusion that instead of mild p53 activity directly influencing cell cycle progression, it actually works by affecting the competitive status of a cell. P53 paired with c-Myc play important roles in cell competition in heterotypic environments. In future experiments it could be analyzed whether in cell fate choices it is either a general stress response where the whole tissue responds or a more localized response where individual cells respond. Further understanding this process can aid in understanding of cancer cells to generate treatments.

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FZD8, a target of p53, promotes bone metastasis in prostate cancer by activating canonical Wnt/β-catenin signaling

In many types of cancers, unregulated cellular proliferation is caused by activating mutations of genes involved in signal transduction pathways. For example, in canonical Wnt signaling, residing proto-oncogenes code for 𝛃-catenin (a transcription factor) and FZD8 (a receptor). Conversely, cellular proliferation can be inhibited by tumor suppressor genes. One such gene is p53 (codes for a transcription factor), which has multiple functions such as inducing growth arrest, DNA repair, and apoptosis. Consequently, mutation of this gene would result in the promotion of cancer. Thus, it is important to investigate the effects of these mutations in order to better understand cancer. In one study, Li et al. manipulated FZD8 expression to examine the resulting Wnt signaling activation, its effects on prostate cancer, and how it would affect bone metastasis.

Compared to other FZDs, FZD8 was significantly more upregulated. This result built off their hypothesis that FZD8 expression would lead to PCa progression and bone metastasis. In addition, such upregulation could lead to enhanced migration of invasion of PCa cells, while reduction of expression would reduce such effects. FZD8 was found to promote the stem-cell phenotypes in PCa cells. Since cancer stem cells (CSCs) are critical to tumor growth and metastasis, this augments the role of FZD8 on metastasizing ability. In relation to the Wnt pathway, their results confirmed that FZD8 contributes to the activation of Wnt signaling in PCa. FZD8 overexpression decreased phosphorylation of 𝛃-catenin, allowing for 𝛃-catenin stability. Thus, Wnt signaling was critical toward promoting PCa metastasis and inducing CSC development. In relation to p53, they found that FZD8 protein level was downregulated when wild-type p53 was transduced into PCa cells. Conversely, FZD8 mRNA expression was upregulated when p53 was silenced in PCa cells, indicating a regulatory function of p53 on FZD8.

Overall, these results suggest that FZD8 expression is associated with PCa progression. The findings of this study indicate how overexpression of oncogenes (such as FZD8) or silencing of TSGs (such as p53) can promote cancerous behavior.

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Dec. 1 Presentations

The lauric acid-activated signaling prompts apoptosis in cancer cells

Lauric Acid (LA) COOH-(CH2)10-CH3 is a saturated medium chain acid found commonly in foods such as plant oils and breast milk. Lauric acid has been linked to many different health benefits. One primary health benefit of LA is its ability to induce apoptosis in cancer cells. It is believed that is done by down regulation of EGFR signaling which is crucial in cancer cell survival. By down regulating EGFR through phosphorylation, it induces apoptosis in the cancer cells. EGFR is an oncogene that is often found in many types of cancer. EGFR is a wt signal pathway. Further research is needed in order to better understand other potential antiproliferative properties LA has. In this study novel information was found in LA and the creation stress fiber formations in cancer cells.

This finding is very important in determining the therapeutic properties of LA and its potential use in cancer treatment and prevention, particularly in both breast and colon cancers. To test the impacts of LA on cancer cells SkBr breast cancer cells were obtained and treated with, for 4 hours, in 100 microM of LA. Another sample was treated with 100 microM of LA and 10microM of a ROCK inhibitor. The group that was treated with the combination developed stress fibers. The Stress fibers then trigger p21^Cip/WAF1_ apoptosis in prostate cancer cells. Since ROCKís role is the regulation of stress fibers and it was inhibited, the research suggests that LA may induce the creation of stress fibers. Creating stress fibers is another trait that induces cell apoptosis in cancer cells along with EGFRwt down regulation.

There were many tests conducted through out the study to determine LA and its ability to induce apoptosis. There was a Chromatin immunoreaction assay, western blot, ROS production, Phalloidin staining, and TUNEL assay. The results were analyzed for statistical significance in ANOVA. The p value determined there was a statistical significance between cancer cells treated with LA compared to controls. More tests need to be conducted on the stress fiber induction and its potential uses in cancer treatment. LA continues to be researched for its potential uses in cancer treatment and prevention.

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