| 2015 | 2014 | 2012 | 2011 |
Pancreatic cancer has the fourth highest mortality rate of cancers in the United States. This cancer is usually caught at later stages, which is one of the reasons that it is a leader in especially malignant cancers. Pancreatic cancer does not respond well to chemotherapy or radiation. ATDC, a gene in the TRIM29 family, is seen in many parts of the body, functioning normally in development and growth. During research in pancreatic cancer, the ATDC protein has been found to be overexpressed in pancreatic cancer and pancreatic precursor cells.
In trying to find out why ATDC proliferation is so great, researchers looked at different signaling pathways. It was found that with an increase of ATDC, an increase in B-catenin in the Wnt pathway was found. When B-catenin is active it moves from the cytoplasm and into the nucleus, where it binds to transcription factors and begins transcription. It is reasonable to assert the idea that ATDC influences B-catenin and activates the target gene, in this case pancreatic cancer cells.
The correlation between the increased presence of ATDC increased the amount of B-catenin was corroborated when researchers silenced ATDC and found that the Wnt pathway functioned in a normal way, with the destruction complex phosphorylating B-catenin and subsequent degradation of the B-catenin. Pancreatic cancer can result in mutations within the destruction complex in the Wnt pathway or other point mutations, such as mutations in the oncogene K-ras, but 65% of pancreatic cancers have an overexpression of B-catenin, both in the cytoplasm and in the nucleus.
Links related to the paper:
The Many Ways of Wnt in Cancer
Progression Model for Pancreatic Cancer
In the 2016 study by Qian-Qian Liu, et al., the researchersí main objective was to determine if Oridonin inhibited pancreatic cancer cells by suppressing the canonical Wnt/ β-catenin signaling pathway. Oridonin (ORI) is a tetracyclin dterpenoid compound that is extracted from the traditional Chinese medicine Rabdosia rubescens. It is primarily used for its anti-inflammatory, antibacterial and antitumor effects, and is known to inhibit the development of various types of cancers by being an effective anti-tumor medicine with little to no toxic effects. The researchers focused on identifying if ORI can suppress the migration of pancreatic cancer even though the mechanisms in which ORI inhibits tumors are still unknown. The researchers aimed at identifying if ORI would suppress the migration of pancreatic cancer through several different assays. They utilized CCK-8 (Cell count kit) assays to determine cell viability by reporting the survival rate as a percentage whilst exposed to different ORI concentrations over different exposure periods. They did this by using different pancreatic cancer cell lines (Axpc1, Bxpc3, Panc1, and SW1990) and observed that the cell line SW1990 was the last sensitive to ORI. Therefore, they decided to focus on that cell line throughout the rest of the experiment.
They compared SW1990 cells treated with 7 and 15 μM ORI to the control group through a wound healing and a transwell assay. Both assays supported that ORI inhibited cell migration. Moreover, the researchers also wanted to examine ORIís effects on tumor metastatic formation by calculating the relative concentrations of mesenchymal molecular markers (such as snail, slug, and Vimentin), and also epithelial-related markers (such as E-cadherin). They did this by conducting a mice model by injective SW1990 cells into nude mice intraperitoneally. Through PCR immunofluorescence of EMT bio markers, it was seen that the expression of mesenchymal molecular markers decreased, and expression of epithelial-related markers such as E-cadherin increased.
Additionally, a Luciferase assay and western blots were done to see ORIís effect on β-catenin and GSK3β concentrations. ORI can decrease the β-catenin protein level not only in the nucleus but also in the cytoplasm and the whole cell after treatment with ORI, and GSK3B was increased in the ORI treatment group. Consequently, there is less β-catenin and higher concentration of the destruction complex that degrades β-catenin.
After supporting that ORI can suppress the Wnt pathway, the above conditions where replicated except that the researchers added CHIIR. They saw that it could override the effects of Ori in SW1990 cells. When ORI was tested for migration: more migration when treated with 2 uM of CHIR, and as well as the number of invasive cells increased. Conclusively, it is supported that ORI inhibits pancreatic cancer cell migration and epithelial-mesenchymal transition by suppressing Wnt/ β-catenin signaling pathway.
Links related to the paper:
Overview of the Wnt/ β-catenin signaling pathway
Wnt/β-catenin signaling pathway explained
More information on Oridonin 1
More information on Oridonin 2
Other Research on Oridonin related to Cancer
Acetylation of the C-terminal domain (CTD) is a form of post-translational modification of both histones and non-histone proteins. The interaction between SET (an oncoprotein) and P53 (tumor protein) is dependent on whether the CTD is acetylated or not by CREB-binding protein (CBP). P53 plays a role in apoptosis, genomic stability, and inhibition of angiogenesis (development of new blood vessels). When P53 is bound to SET, inhibition of P53 is observed and cell proliferation is enabled. Parallels to the interaction between Set and the CTD of P53 were also observed between VPRBP, DAXX, and PELP1 where the formation of the complex was dependent on lysine rich domains (KRD). These lysine rich domains have an overall positive charge which allows the recruitment and interaction with the acidic, and negatively charged, domain of SET and the proteins mentioned above.
In vitro analysis of HCT116 cells demonstrated that SET had no effect on DNA stability but did influence histone modification at P53 target promoters. When SET was suppressed acetylation of H3K27 and H3K18 at the promoter of p21 and PUMA in HCT116. In vivo experimentation using P53 KQ/KQ mutant mice and SET mutant mice demonstrated that SET is a key regulator for P53. In short by controlling P53 acetylation you can affect the interaction between the oncoprotein SET and P53 and promote P53 function.
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Reduction of breast cancer-associated gene 1 (BRCA1) expression has been shown in sporadic (meaning somatic not germline mutation) breast cancers. Most often this is not associated with an actual mutation in the BRCA1 gene or molecular modification such as methylation of its promoter. The mechanism by which BRCA1 reduction could be causally linked to cancerous cells is unclear. The authors were interested in the regulation of BRCA1 that may lead to its reduction rather than the effects of BRCA1ís reduction.
BRCA1 has two promoters: α and β. By attaching these promoters and variable amounts of the sequences around them to luciferase, a reporter enzyme whose catalytic activity can be monitored as an indication of the level of transcription, the authors found that luciferase attached to a small segment containing only the α promoter resulted in the most luciferase activity. This indicated that the β promoter and surrounding sequences had negative elements, like areas where repressors of transcription could bind. They used the small construct containing only the α promoter as their reporting construct to test 92 transcription factors that were well known to regulate BRCA1 expression. They found several that significantly upregulate and down regulate BRCA1. However the authors chose to focus on factors that upregulate BRCA1 expression, and the transcription factor with the greatest effect was Yin-Yang1 (YY1.) It is a zinc-finger protein in the Gli-Krueppel class. YY1 has been shown to have conflicting oncogenic and tumor suppressing effects. It can up regulate oncogenes like c-myc and ErbB2, and reduce the stability of a tumor suppressor like p53. However, it can also bind and inhibit c-Myc and positively regulate tumor suppressor genes like p53 and p73. Itís effects are contextual and tissue specific.
Authors first explored whether YY1 was directly or indirectly affecting BRCA1 expression. Since BRCA1 is known to increase and decrease through phases of the cell cycle, authors showed that the effects of overexpression of YY1 on cell cycle were not responsible for the increase; they showed YY1 arrested cells in a phase that usually correlates low BRCA1 expression. They then sought to prove YY1 directly bound BRCA1ís promoter, which they showed by using chromatin immunoprecipitation which showed them it did directly bind the promoter. They identified the region where YY1 bound the BRCA1 promoter with serial deletions, when the region was deleted a reduction in BRCA1 expression was observed. To further confirm that BRCA1 and YY1 had a positive correlation in vivo, researchers took tissue samples known to have differential expression of BRCA in vivo, specifically differences between lactating and nonlactating mammary glands. The observations supported the previous data of a positive correlation between YY1 and BRCA1 expression.
Next, the authors looked at YY1 as it relates to cancer. They found by testing cell lines that are cancerous in vivo that addition of YY1 reduced cell proliferation, the number, and size of cell colonies. However, when YY1 was added to cell lines that had been treated with BRCA1 shRNAi, the reduction phenotype was not observed, suggesting YY1 reduction phenotype was a consequence of its interaction with BRCA1. Researchers also found that overexpression of YY1 could eliminate or reduce tumor formation. The authors next confirmed that human sporadic breast cancers have lower expression of YY1, providing insight into the mechanism of BRCA1 expression reduction in humans. From this they were able to conclude that reduction or loss of YY1 function could be one of the factors that leads to sporadic breast cancer, and that YY1 has another function as a tumor suppressor.
Links related to the paper:
explanation of luciferase assay
explanation of immunoprecipitation
functions of c-Myc as an oncogene
example of some of the negative elements in BRCA1 promoter/upstream sequence
The BCL-2 protein family is known as an important gatekeeper to the apoptotic (programmed cell death) response. The dynamics of how this family of proteins interacts remains controversial. Within the BCL-2 family of proteins there are anti-apoptotic proteins (ones that attempt to prevent cell death) as well as pro-apoptotic proteins (which try to initiate the cell death process). Within the group of pro-apoptotic proteins there are BH3-only activators that can activate the other subgroup of pro-apoptotic effectors (BAX & BAK).
Activated BAX and BAK initiate a membrane pore to be created within the mitochondria (MOMP process), inducing the release of cytochrome c, which is the next step in the cell death process.
These researchers wanted to understand the exact nature of the protein-protein interactions (between BCL-2 with BH3-only activators as well as with BAX and BAK). They identified two 'modes' by with pro-survival BCL-2 proteins can block MOMP (mitochondrial outer membrane permeabilization).
'Mode 1' occurs when BCL-2 proteins sequester the direct-activator BH3-only proteins (so that they cannot activate BAX or BAK). 'Mode 2' occurs when BCL-2 proteins bind directly to BAX and BAX, preventing their mitochondrial fusion.
Links related to the paper:
The BCL-2 Family: A Balance Between Pro- and Anti-apoptotic Protein Expression
How do BCL-2 Proteins Induce Mitochondrial Permeabilization?
Overview of MOMP (Mitochondrial Outer Membrane Permeabilization)
Recently, studies have shown that metformin, an anti-diabetic drug, can indirectly decrease tumor proliferation. In the past, metformin has been used to treat type 2 diabetes. It acts by activating a protein kinase (AMPK) that inhibits hepatic gluconeogenesis. AMPK regulates the phosphorylation of p27 and can also activate forkhead transcription factor (FOXO) proteins under certain conditions. AMPK activation also results in decreased blood glucose and insulin levels. The lower insulin levels activate insulin pathways such as PI3K/Akt/mTOR and MEK/ERK1/2 that lead to decreased tumor proliferation. Metformin inhibits mTOR activity to inhibit cancer growth. However, the mechanisms underlying the effects of metformin are not well understood. So, Quieroz and her team treated MCF-7 (breast cancer) cells with different concentrations of metformin for 24, 48, and 72 hours in order to observe and quantify its effects.
BrdU and MTT assays demonstrated that metformin effected proliferation based on both its time of treatment and the concentration of the treatment. Flow cytometry was used as a tool to analyze where in the cell cycle the cells were at given time intervals. It was also used to observe markers of apoptosis, necrosis, and oxidative stressors. Subjection to metformin caused cells to arrest in the G0-G1 phase, and also resulted in a significant increase in necrosis and apoptosis. Real time reverse transcription PCR and and western blotting were used in order to investigate the genes and proteins expressed in these cells, respectively. Metformin was found to decrease mRNA levels of cyclin D1 and Bcl-2 genes, and increase mRNA levels of p27 and Bax genes.
Additionally, the research team wanted to determine if oxidative stress was a part of the underlying anti-proliferative mechanism of metformin. They found that cells had increased viability when treated cells with metformin in combination with a superoxide dismutase (SOD, an antioxidant), as opposed to metformin alone. These results indicate that oxidative stress contributes to its anti-proliferative effects.
This study demonstrated that metformin inhibits MCF-7 cell proliferation by controlling the cell cycle, up-regulating tumor suppressor genes, and inducing cell death via increased oxidative stress. AMPK and FOXO3a genes express proteins vital to the anti-proliferative effects seen from metformin exposure. These findings reinforce the possibility of using metformin as an effective treatment for breast cancer, and potentially other cancers as well.
Links related to the paper:
Metformin and reduced risk of cancer in diabetic patients.
The Ras-ERK and PI3K-mTOR Pathways: Cross-talk and Compensation
Caspases are enzymes that perform proteolysis, the catabolic breakdown of proteins. The role of caspases has been extensively studied in apoptosis, the process of programmed cell death. C. elegans, a classic model organism, contains four caspase genes within its genome: ced-3, csp-1, csp-2, and csp-3. The vast majority of previous research on these genes demonstrates that ced-3 promotes apoptosis. However, through RNAi ìknockdownî experiments and extrachromosomal arrays (which generate an extremely large amount of mutant combinations), Dennings et. al discovered that csp-1 also encodes a caspase that promotes apoptosis.
csp-1, a maternal effect gene, has the ability to encode three potential isoforms: csp-1A, csp-1B, and csp-1C, of which only csp-1A contains a lengthy prodomain. Furthermore, Dennings et. al determined that the isoform csp-1B encodes a pro-apoptotic caspase; the caspaseís activity is dependent on the location of a cysteine within the active site. The cells that were 'killed' by csp-1B (primarily neurons) portray typical apoptotic phenotypes, including chromatin condensation and cytoplasmic contraction. The apoptotic activity of the caspase encoded for by csp-1B occurs independently of the egl-1 pathway.
Dennings et. al also discovered that caspases are not absolutely essential for apoptosis; cells can be eliminated through either phagocytic or ìcell-sheddingî (also known as extrusion) mechanisms even when all four caspase genes are mutated. This apoptotic pathway occurs at a slower rate, however. Thus, various subsets of cells possess different sensitivities to certain pro-apoptotic signals. The range in apoptotic pathways ensures complete cell death for cells ìfatedî to die, regardless of the existing environmental conditions.
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Caspase Functions in Cell Death and Disease
Scientists have already discovered the pathway that most cells in C. elegans use for programmed cell death. However, there are a couple of important areas in which the regular pathway doesn't seem to apply. One of them is the tail-spike. The egl-1 protein that plays a huge role in the regular pathway seems to only have a peripheral role in tail-spike cell death.
Through a series of experiments, Chiorazzi, et. al discovered that the DRE-1 gene primarily regulates cell death. Through an RNAi experiment, they determined that dre-1 interacts with several other proteins like skr-1 and cel-1 in an SCF complex. By studying mutations of the DRE-1 gene, they found that dre-1 functions in parallel to egl-1 and binds to CED-9.
Because they found a relationship to CED-9 in cell apoptosis and knew that BCL2 proteins like ced-9 are often overexpressed in human blood cancers like lymphoma, Chiorazzi et. al hypothesized that an F-box protein like dre-1 might control BCL2 function in lymphoma. Through a series of experiments, they found that FBXO10 (an F-box protein in humans) directly plays a role in BCL2 protein stability. They found that FBXO10 is a component of an ubiquitin ligase that can target BCL2 protein for degradation. Through a series of protein expression studies, they found that it functions to promote cell death, just like DRE-1 in C. elegans.
Overall, this study highlighted a previously unknown mechanism of cell death regulation in C. elegans. Potentially, this information could be used to gain more insight into the regulators of tumorgenesis and developmental cell death.
Links related to the paper:
Programmed Cell Death - Figure 3 is especially helpful
SCF complex and ubiquitin ligases
Overview of known C. elegans cell death pathway
While positive regulation of the RTK cell signaling pathway is often focused upon, there are also inhibitors of the pathway, one such being the Sprouty proteins. The proteins regulate development in angiogenesis, the limbs, wings, and eyes among other systems. Shin et al. focused on elaborating the role of Spry1 and Spry2 in the development of the mouse eye.
After creating a gain of function mutant for both Spry2 and Spry1, many aspects of the lens such as cell size were assessed to observe the inhibitory effects of spry in proliferation and fiber cell differentiation. To understand the molecular mechanisms involved in the effects on the lens cells, the amount and presence of ERK1/2 was measured and the cells were treated with FGF and EGF to determine if any ERK1/2 phosphorylation occurred. Spry regulates phosphorylation of the kinase ERK1/2 which leads to the inhibitory effects of the RTK signaling pathway. Thus downstream effects such as cell proliferation are affected.
Sprouty proteins are extremely important in many areas of development and play an important role in the regulation of RTK cell signaling pathways as well as in cell proliferation and fiber cell differentiation. Inhibitors are just as important when regulating cell signaling pathways. Further exploration into the use of inhibitors such as sprouty to reduce unwanted cell proliferation may prove informative.
Links related to the paper:
RTK Cell Signaling Pathway Overview
The ERK Signal Transduction Pathway
Sprouty proteins: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling
Negative Regulation of Receptor Tyrosine Kinase (RTK) Signaling: A Developing Field
Sprouty proteins: modified modulators, matchmakers or missing links?
In the 2015 study by Yannan Fan, et al., the researchers were ultimately trying to find how sensitive the development of animals is to a fluctuation in RTK signaling. In this experiment the researchers made transgenic mice and used Cre-Lox technology to change the expression time, the quantity of expression, and the expression locations of Met. They found that one of the things affected by ubiquitous expression of Met, was the migration of myoblasts. The researches then focused on this event and did several experiments in order to figure out what was going on at a molecular level.
There are basically two parts of myoblast migration. First, the myoblast moves, and second, it arrives at the limb mesenchyme. The researchers sought out to determine if the problem was due to the enhanced Met signaling in the myoblasts or the ectopic Met signaling in limb mesenchyme. What they found was that increased Met signaling in the myoblasts themselves had no effect on their migration. Since the myoblasts withstood a change in the level of expression of Met they were termed robust. Further research where Met signaling was ectopically expressed in limb mesenchyme did demonstrate a problem with the myoblast migration. There are two reasons for this, one is a down regulation of two morphogens that otherwise create a gradient, and the other is a decrease in the bioavailability of HGF, the ligand.
The researchers demonstrated that enhanced activation of Met signaling where the signal is endogenously expressed does little to effect the overall phenotype. This change may be negligible, or it may be checked by other signals the cell receives. However, something that is sensitive and not robust, such as limb mesenchyme, can have a complete disruption of normal functioning if Met signaling is messed with in any amount.
Links related to the paper:
An overview of the c-Met signaling pathway
Video Clip - Helps to explain Cre-Lox Recombination
Spatial regulation of receptor tyrosine kinases in development and cancer
CELL SIGNALLING DYNAMICS IN TIME AND SPACE
Calpains are intracellular cysteine proteases that are calcium-dependent and control the limited cleavage of proteins in order to regulate signal transduction, cell proliferation, apoptosis, and cell motility. When these processes are not properly regulated, cancer, Alzheimer’s, and many other severe diseases may develop. Through knockout experiments with mice, it was found that Calpain2 is necessary for cell viability. It was concluded that both Calpain1 and Calpain2 are maternal proteins (maternal effect genes) expressed throughout development.
This study explores the function and expression of Calpain2 and found that it is essential for mesoderm migration and convergent extension during gastrulation and neurulation by regulating cell shape and cell polarity. Convergent extension occurs when the mesodermal cells converge toward the dorsal midline and elongate along the anterior-posterior axis during gastrulation and the spinal cord and posterior hindbrain converge and extend during neurulation. This process is regulated by the non-canonical Wnt pathway, otherwise known as the planar cell polarity (PCP) pathway.
They discovered that Calpain2 is activated in response to Wnt5a and is present in the Wnt pathway. Calpain was manipulated in this experiment to observe the effects during development of Xenopus when inhibited. They found defects in mesodermal morphogenesis as well as defects in neural tube closure. This protease needs to be precisely regulated because overexpression or inhibition will block convergent extension. This research is important to determine if Calpain is a major factor in development, and what can be done to rescue this protease if mutated or inhibited, to prevent severe birth defects as well as determining its specific function with regards to morphogenetic movements.
Links related to the paper:
Wnt/Ca2+ Signaling Pathway: a brief overview
Calpain 2 expression pattern and sub-cellular localization during mouse embryogenesis
Calpains expression during Xenopus laevis development
Calpain: A Protease in Search of a Function?
The Calpain System Is Involved in the Constitutive Regulation of B-Catenin Signaling Functions
Neural crest cells are highly dynamic and migratory cells that require canonical Wnt signaling for differentiation to their specific end locations. Neural crest cells delaminate from the neural tube to migrate and differentiate into a variety of cell types including: glial cells, smooth muscle cells, craniofacial cartilage and bone, pigment cells, amongst others. It is currently known that the Wnt signaling pathway is sufficient to regulate and induce the movement of these cells through either inhibition or activation at certain times in embryonic development. More specifically, the canonical Wnt signaling pathway has a broad range of functions in the pre and post – induction stages of the NC development.
In this study, Maj and colleagues used inducing and inhibiting agents at pre-migratory and migratory stages to further evaluate and dissect the role that canonical Wnt signaling as in NC migration. In both stages of embryonic development, they found that there needs to be a close regulation of Wnt signaling to allow for proper NC cell migration. The scientists found that the permanent up-regulation of the canonical Wnt signaling pathway (the increase of B-catenin levels and increase in transcription) produced severe NC migration defects. This indicates that the functions of the Wnt signaling in these cells are tissue autonomous. The effects of the up-regulation were similar in both pre-migratory and migratory stages. Incidentally, the down regulation of the same canonical Wnt signaling pathway had similar effects.
Overall, the data from this study suggests that Wnt needs to be activated to allow for delamination, but it also needs to be down regulated to allow for the proper migration of the neural crest cells to their proper locations.
Links related to the paper:
Wnt signaling/role of B-catenin - This is a video discussing the basics of the Wnt signaling pathway and the role that B-catenin has in transcription.
Explanation of NC cells and where they are derived
Wnt/β-Catenin Signaling in Vertebrate Posterior Neural Development.
Wnt–frizzled signaling in neural crest formation