"Read More About It" Pages - Biology 376 - Fall 2014

Dec. 12, 2014 Presentations

Inhibition of Myc family proteins eradicates KRas-driven lung cancer in mice

Myc family protiens are responsible for many critical steps in cell growth, proliferation and apoptosis. Myc, and more specifically c-myc is degregulated in many, if not all cancers, making it a useful target for therapy and future research. Ideal cancer drugs should target a function that is essential for tumor growth and survival, but that also doesn’t damage healthy tissues. A common pitfall of current treatments is the eventual resistance to the drug given, and the regeneration of a stronger, resistant tumor. Myc is found to function in aiding the expression of many different genes that together help promote somatic cell proliferation. It has been shown that myc inhibition using a dominant negative Myc mutant called Omomyc can greatly regress tumors with only slight harmful yet reversible effects on healthy tissue. This study shows that if omomyc is given in low doses in regular intervals over a prolonged period of time, it can greatly reduce the adverse effects of the treatment as well as has shown to indefinitely prevent new tumors from forming.

This suggests that Myc inhibition in this fashion, does not create mutant clones that cause resistance. This is even true in the most aggressive p53 deficient cancers. In a normal situation (non-cancer) when c-Myc is overexpressed p53 is induced to let the cell know that a crisis has occurred and starts the journey to apoptosis. In this way p53 commonly regulates c-myc. When p53 is not working correctly, it does not respond to c-myc overexpression and a decreased apoptotic response is observed.

In order to function as an agent in proliferation, as well as oncogenetic activities c-myc dimerizes with Max. Myc inhibition is achieved by blocking this interaction with Omomyc. Myc function is not replaced by any other pathway or mechanism once it is inhibited, making a very promising target for treatment. The actual mechanism for how omomyc promotes cancer cell death is still not well understood, but is a possible plug for future research.

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The Core Apoptotic Executioner Proteins CED-3 and CED-4 Promote Initiation of Neuronal Regeneration in Caenorhabditis elegans

The repair and regrowth of neurons damaged by injury, disease or surgery is a primary goal of regenerative medicine. Improvements in laser technology now allow individual axons to be cut and analyzed for regrowth in vivo.

In the present study, Pinan-Luccare et al. found that the core apoptotic proteins, CED-3 and CED-4, promote the regeneration of severed neurons in C. elegans. Mutants lacking ced-3 displayed impaired regenerative outgrowth and delayed reconnection of the dissociated ends. CED-4, the activator of CED-3, was also found to be necessary for the initiation of the regrowth of damaged neurons. However the other regulators of apoptosis, CED-9 and CED-13, were not required for regrowth.

CED-3 caspase was found to function in a calreticulin, calcium-dependent pathway that requires calcium signaling and functional calreticulin gene, crt-1. Thus the core apoptotic effector proteins function in a novel pathway that promotes neuronal regeneration. The results draw into question the use of anti-caspase therapy to limit nerve-loss following nerve injury.

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The clinicopathological significance and relationship of Gli1, MDM2 and p53 expression in resectable pancreatic cancer

Pancreatic cancer has a 5-year survival rate of <5%, due to the fact that symptoms do not present themselves until advanced stages and the cancer spreads rapidly. Because of it’s poor prognosis, W Sheng et al aims to investigate the relationship between the expression of Gli1, MDM2, and p53 in pancreatic cancer, hoping to further investigate their involvement in the aggressive behaviors of pancreatic cancer.

Previous studies have shown that all 3 play a part in the initiation and progression of cancers. Over expression of Gli1 (a transcription factor), activated by the Sonic hedgehog (Shh) signaling pathway, can result in tumor formation and a mutation in the P53 tumor suppressor gene results in reduced apoptosis and excessive cell proliferation. Sonic hedgehog is also known to induce MDM2, which is linked to p53 in an autoregulatory negative feedback, where MDM2 is the product of p53 and MDM2 in return inhibits p53.

After performing various knockdown tests of Gli1, MDM2, and p53 using siRNAs, immunohistochemistry assays, immunoblot assays, and PCR on both mutated p53 cells and normal p53 cells, Sheng et al found that patients who have Gli1 expression only or had expression of both Gli1 and MDM2 had a lower survival rate than patients who have negative expression. Sheng et al also observed that mutant p53, and overexpression of both Gli1 and MDM2 contributes to the advancement of pancreatic cancer. Although they found a positive relationship between Gli1 and MDM2, their effect on p53 was dependent on whether p53 was mutant or wild type.

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Testing Models of the APC Tumor Suppressor/B-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

Wnt signaling as we have learned in class is widely utilized in many cell signaling pathways mainly resulting in transcription for cell proliferation. When parts of this pathway become mutated, a common result is the formation of cancer due to extra signals for proliferation.

In this study, the "destruction complex" was under scrutiny, specifically the tumor suppressor adenomatous polyposis coli (APC) component. Other components are Axin, GSK3, and CK1 with Axin interacting with Disheveled and GSK3 and CK1 seeming to be involved in phosphorylation of APC and β-catenin. Specific binding sites displaying 15 amino acid repeats (15Rs) and 20 amino acid repeats (20Rs) were analyzed and mutated to observe their effect on β-catenin accumulation, restoration to wild type levels, or no effect at all. To analyze APC, Drosophila APC genes APC1 and APC2 were hypothesized for use because vertebrates contained far too many APC's and APC binding sites to manipulate, however only APC2 was investigated. Mutants created were: APC2Δ20R2 meaning only the second amino acid repeat was knocked out, APC2Δ20R1, R3-R5 meaning this mutant only had the second amino acid repeat described above, APC215Rs, 20R1, R4,R5 meaning all the 15Rs were knocked out and only the 20R2&3 remained.

From the results it seemed the 20R2, which did not have the ability to bind β-catenin, still rescued any mutation and was suggested to be vital to APC functionality. The 20Rs had more positive rescue on β-catenin levels than 15Rs. Even levels of β-catenin were reduced when all sites from APC were eliminated suggesting actual binding to APC was not necessary for β-catenin destruction and even at times it seemed β-catenin was being retained in the cytoplasm, not letting it enter the nucleus.

Overall goal of this research was not directly stated in the paper but it would seem advantageous to understand the destruction complex in order to possibly control levels of β-catenin that is promoting cancer. Understanding the key binding sites and proteins may help in new drug treatments toward stopping tumorigenesis.

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Dec. 8, 2014 Presentations

Far upstream element-binding protein-1, a novel caspase substrate, acts as a cross-talker between apoptosis and the c-myc oncogene

Apoptosis is a key mechanisms to maintaining homeostasis and helps dismiss damaged or unwanted cells. In addition to being a proto-oncogene, c-Myc also acts as a tumor suppressor gene, and resides within the Myc family of short-lived nuclear phosphoproteins. These proteins are multi-functional, and are associated with cell growth, cell cycle progression, apoptosis, and cellular differentiation via transcriptional modulation. Specifically, c-Myc encodes for a major chunk of the basic helix-loop-helix leucine zipper family of transcription factors, and is nearly only active when a specific protein that bind upstream of the c-Myc promoter; appropriately named 'Far Upstream Element-Binding Proteins' (FBP). Low levels of c-Myc mRNA and protein are found in normal proliferating cells, and slight deviations from this optimal concentration lead to either cell proliferation or apoptosis. This study looks at when and where this transition from oncogenic to tumor suppressant occurs.

Previous studies have shown this proto-oncogene to also play a role in the induction of apoptosis by means of Bax oligomerization and the 'caspase feedback amplification loop' (Cao et al, 2008). This feedback amplification loop is essentially the point at which apoptotic events are irreversible. The activation of initiator caspases lead to a self-promoting mechanism that leads to the degredation of cytochrome c in the mitochondria, as well as other interactions involving their target substrates, such as FBP-1 (Chen et al, 2005). I addition, this interaction of FBP-1 with caspases appear to be the final nail in the coffin for the cells fate (pun intended).

The present study assists us on the understanding of the sequence of events leading to apoptosis by means of caspase monitoring. The results show FBP-1 as a candidate substrate for the effector caspase-7, and thus, when activated by apoptotic stimuli, caspase-7 will cleave FBP-1 and lead to the down regulation of c-myc. For that reason, the downregulation of the FBP-1 may function as the 'switch off' mechanism for the caspase feedback amplification loop, as well as c-Myc's proliferation signalling function.

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Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Some antibiotics acting as polyether ionophores have been used in selective cancer stem cell inhibition, but have also been found to have some anticancer effects. With increasing amount of studies done on Wnt/β-catenin signaling pathways and the connection to cancer activation, this study aims to characterize salinomycin as an inhibitor of the pathway by means of ionic changes that interfere with LRP6 phosphorylation.

LRP6, or lipoprotein receptor related protein 6, is a necessary co-receptor in the Wnt signaling pathway that interacts with Dickkopf-1 (DKK-1) for activation. The experiments done by Lu et al. have found salinomycin as an antagonist promoting the degredation of β-catenin and the downregulation of Wnt target genes contributing to cancer. In this study, chronic lymphocytic leukemia (CLL) is examined due to its prevalence in the Western world, and the actions of salinomycin have been directed toward these cells. Even at subnanomolar concentrations, salinomycin was found to be toxic selectively to primary CLL cells, but still suppressed Wnt-induced LRP6 phosphorylation.

There is so much potential in targeting LRP6 for cancer cell therapy through the use of drugs that alter phosphorylation and stability. Studying the mechanism of ion exchange and its effect on the canonical Wnt pathway may help in the development of drugs and antibiotics that have potential therapeutic use in cancer.

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BRCA1 and p53 regulate critical prostate cancer pathways

Many anticancer drugs rely on the presence of p53 wild-type in tumorous cells, such as Doxorubicin. The expected result for these drugs is p53-dependent apoptosis or senescence. If p53 is not present or expresses p53 (mutant) there is increased resistance in chemotherapy. There is high frequency of these mutations in human tumor cells thus, research concerning p53 is important.

Breast cancer susceptibility gene 1 (BRCA1) is a genetic predisposition for breast, ovarian and in this case prostate cancer (PCa). Previously the DeLuca group determined that GADD153, promoter protein in the G2/M DNA damage signaling pathway, increases apoptosis and cell cycle arrest in BRCA1 over-expressing cells; inducing doxorubicin sensitivity.

In this experiment the DeLuca group investigates the effect of p53 over GADD153 regulation by BRCA1 during the DNA damage response—specifically in PCa LNCaP cells that express p53 wild-type. What was found is BRCA1 targets GADD153 promoter and during DNA damage response increases transcription. It was also shown that p53 abolishes GADD153 induction from DNA damage. The explanation for this may be due to BRCA1 being repressed by p53. BRCA1 protein binds and regulates GADD153 promoter, independently of p53 presence, but GADD153 induction from DNA damage is lost in p53 expressing cells due to p53 repression of GADD153 and BRCA1. Both BRCA1 and p53 regulate several pathways in PCa cells especially in DNA damage, EMT and Hedgehog pathway. The importance of this study shows that BRCA1 and p53 and extremely important transcription regulators in PCa.

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Dec. 3, 2014 Presentations

Autophagy genes function in apoptotic cell corpse clearance during C. elegans embryonic development

During normal development, many cells undergo apoptosis and it is important for these cells’ corpses to be removed from the embryo following this programmed cell death. This regulated process involves the display of an “eat me” signal which tells neighboring cells to consume and degrade the corpse.

Many studies involving chick and C. elegans embryos suggest that autophagy proteins may be essential for corpse clearance during programmed cell death. Further support of this is seen in Huang et al (2013) where an increased number of apoptotic cell corpses are detected in C. elegans embryos with mutations in autophagy genes (such as bec-1). This difference can be explained by a delay in corpse clearance, as this paper demonstrated.

Results suggested that bec-1, and other autophagy genes, may function in both apoptotic cell corpse engulfment and degradation during C. elegans embryonic development.

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C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions.

Apoptosis, programmed cell death, occurs in multicellular organisms and can be caused by extracellular or intracellular inducers. Cells undergoing apoptosis will undergo morphological changes, which include nuclear fragmentation, bulging of the cell membrane, cell shrinkage, chromatin condensation. Apoptosis is beneficial for maintaining cell homeostasis and for protecting surrounding cells.

Previous studies have shown that this execution of cell suicide helps to regulate the number of available cells. C. elegans undergo apoptosis during early embryonic development, “to endure functional organ architecture and to maintain tissue homeostasis” (Lettre and Hengartner, 2006). During C. elegans development, 131 out of 1090 somatic cells that are formed undergo apoptosis (Sulston and Horvitz, 1977; Sulston et al, 1983). These 131 somatic are not necessary for the viability of the nematodes. It is important to be able to be able to follow the life and death sequence of the cells and how it is able to occur. Three key genes, which are necessary for programmed cell death in development (apoptosis), have been identified in C. elegans: ced-3, ced-4, and egl-1 (ced denotes cell death abnormal, while egl stands for EGg laying defective). Ced-3, ced-4 and egl-1 are all pro-apoptotic genes and if a loss-of-function mutation occurs in any of these genes then the cell death of most or all the cells is sufficiently repressed. Developmental apoptosis is also blocked by ced-9. Constitutive germ cell apoptosis (CGCA) is dependent on ced-4 and ced-3 and is blocked by ced-9 (Schertel and Conradt). CGCA however is not dependent on egl-1.

In humans the retinoblastoma is functionally inactivated in most human solid tumors. The RB has been known to block cell proliferation by binding proteins that will block transcription before the G1- S phase. Similarly to RB gene in humans, C. elegans have orthologs of components of the RB tumor suppressor complex. lin-35, like RB, also has functional role in the differentiation and proliferation of the cell. This article looked at the lin-35 as well as other genes necessary for germ cell apoptosis and how they function. These other genes include dpl-1 DP, efl-1 E2F and efl-2 E2F. lin-35 role in apoptosis has never been studied before. This article showed that lin-35 and dpl-1 DP, efl-1 E2F and efl-2 E2F all play a role in germ cell apoptosis, but lin-35 role was different from the other genes. This article showed that lin-35/Rb inhibits ced-9/ Bcl-2, which would promote CGCA by blocking the anti-apoptotic gene ced-9. Where as dpl-1, efl-2 and efl-1 induce the function of ced-4 and ced-3 downstream of ced-9. Although all of these genes function in the normal C. elegan cells, when the DNA is damaged efl-1 does not play any role in apoptosis in these cells.

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APP Binds DR6 to trigger axon pruning and neuron death via distinct caspases

In a normal system, pruning of axons and neuronal cell death occur naturally to help shape and improve neuronal connections. This occurs all throughout the nervous system during development as well as after injury in adults and is implicated in several neurodegenerative diseases, including Alzheimer's disease. Neuronal development consists of two stages, the development stage and the regressive stage. In the development stage, neurons are generated and axons extend out to form connections. After this, there is what is called a “regressive phase” where only a portion of the neurons made are selected to live while the rest die by apoptosis, and the axon branches are reduced to make the connections more precise. Despite the importance of these processes, very little is known about the mechanisms by which this occurs.

To perform this study, Nikolaev et al. used several different methods. Fluorescence in situ hybridization was used to screen TNF receptors to analyze their involvement in neuronal development and it is from this that DR6 was chosen as a focus. This was due in part to its high expression in dividing neurons in the spinal cord and adjacent ganglia. In order to analyze the specifics of axon pruning, Campenot chambers were used and the axons were stained and observed with TuJ1 immunostain while various trophic factors (TFs) and nerve growth factors (NGFs) were added or removed.

From this experiment, the researchers propose that there is a pathway for cell death and axonal degeneration that follows a 'ligand activation' model. They also suggest that it is caspase-dependent and catalyzed by the binding of a fragment of β-amyloid precursor protein (APP), called the N terminus of APP (N-APP), to death receptor 6 (DR6). They also report that there is a difference in the type of caspase that is involved in the death of the neuronal cell body (caspase 3) and the pruning of the axons (caspase 6).

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Sonic Hedgehog promotes the survival of neural crest cells by limiting apoptosis induced by the dependence receptor CDON during branchial arch development

Dependence receptors are a potential target for cancer treatments and are receptors that initiate apoptosis in the absence of their ligand. By forcing expression of these receptors in tumor cells, apoptosis can be induced. Understanding the natural function and interactions these particular receptors have is therefore important.

Cell-adhesion molecule-related/downregulated by oncogenes (CDO) is a dependence receptor involved in canonical Sonic Hedgehog (SHH) signaling. This specific study sought to understand CDO’s activity in branchial arch formation through in vivo experimentation.

In order to achieve this goal, electroporation was performed on half of chick neural tube to force expression of CDO in this location. Neural tube was utilized as the experimental site because SHH is known to be expressed in the neural tube in a dorsal-ventral concentration gradient. Monitoring of CDO expression using immunofluorescence as well as monitoring of apoptosis through TUNEL staining showed heightened apoptosis in the dorsal region of CDO expressing neural where SHH concentration is low. CDO thus activated apoptosis in the absence of its ligand, SHH.

This mechanism was also present in branchial arch development. SHH secreted by the ventral foregut was responsible for the survival of facial neural crest cells. CDO caused facial neural crest cells forming in the incorrect location to undergo apoptosis.

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β-catenin regulates Pax3 and Cdx2 for caudal neural tube closure and elongation

Neural tube defects are severe birth defects caused by a failure of the neural tube to fully close, including spina bifida and anencephaly. There are several points at which closure happens throughout development, the final being the posterior neural pore (PNP). NTDs may have a genetic component, but only a few genes have been linked to human NTDs. However, in mice, over 200 genes have been identified. Since the mouse is used as a model organism for humans, some of the genes causing the defects in the mice are likely to have a homolog involved in the same process in humans. The better understanding gained about neural tube closure in mice, the better NTDs in humans can be identified early in pregnancy.

Previous studies have shown that there is strong evidence for the non-canonical Wnt, or planar cell polarity (PCP) pathway being involved in the process for neural tube closure (Wallingford, 2006). However, many of the molecules involved in the PCP signaling pathway are also involved in the canonical Wnt signaling pathway (MacDonald et al, 2009). This study aims to analyze the role of the canonical Wnt signaling pathway in neural tube closure. The hypothesis states that canonical Wnt/β-catenin signaling is required for neural tube closure in mice through the regulation of transcription of downstream target genes, including Pax3 and Cdx2.

Using Cre-lox conditional gene targeting, the researcher created a β-catenin conditional knockout (cKO) to analyze the role of the canonical Wnt signaling pathway. The study showed that after 9.5 days of development, which equates to about 22 days in humans, there was defective PNP closure and elongation of the embryos, showing that β-catenin is indeed necessary for PNP closure. In addition, the study shows that Pax3 and Cdx2 are target genes of the canonical Wnt signaling pathway, because levels of Pax3 and Cdx2 were repressed in the β-catenin knockouts. Finally, the study showed that transgenic activation of Pax3 cDNA can rescue the PNP closure defect, but cannot restore Cdx2 levels on its own. Therefore, the study concluded that β-catenin is in fact necessary for caudal neural tube closure and elongation, and that it functions through the transcription of target genes including Pax3 and Cdx2.

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Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse

Multiple studies in the past have looked at the embryonic pattering in different model organisms at the molecular level. Anterior visceral endoderm (AVE) is found to express inhibitors of Wnt/β-catenin pathway. Posterior visceral endoderm (PVE) allows the formation of the gastrula organizer as well as the primitive streak. This consequentially leads to definitive endoderm (DE). A previous study showed that by deleting the β-catenin in epiblast progenitors, there is a loss of DE. This illustrates that Wnt and β-catenin are necessary in the development of mesoderm and endoderm specification.

However, the purpose of Wnt/β-catenin signaling in detail for AVE, PVE, and DE is not yet understood. The studies done by Engert et al discuss the function of this signal pathway by using conditional knockout protocol to see the effects of removing β-catenin resulting from the Sox17 gene. The CKO embryos result in head and tail truncations, causing them to be embryonic lethal.

To perform the study, Engert et al floxed as well as flox deleted β-catenin alleles for Sox17-pos mice. Immunofluorescence methods was performed using various tagging antibodies. Furthermore, tetrapod chimeras were used to observe the rescue of head/tail truncated embryos. Embryos were stained with a β-galactosidase stain, dehydrated via ethanol series, and mounted on slides.

Engert et al discovered that SOX17 was expressed in AVE and PVE on day 6.5 of development, while it was expressed in DE at day 7.5. More importantly, they found that the absence of β-catenin resulted in truncated versions of the neural tube and head, indicating the there was a defect in the organizer tissues (ie. AVE and DE). This suggests that the β-catenin loss results in non-autonomous defects resulting in head and tail truncations; β-catenin is necessary for the AVE and DE formation and consequentially head and neural tube formation. Moreover, they found that the Wnt/βcatenin signaling in Sox17 allows for the specification between De and mesoderm epiblast. CKO on Sox17 embryos resulted in gut defects indicating Sox17 plays a role in the Wnt signaling of a developing mouse.

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Inhibition of Wnt/β-catenin pathway by Dikkopf-1 affects midfacial morphogenesis in chick embryo

The Wnt pathway is a very important pathway involved in development of embryos. Recent studies regarding the Wnt pathway have shown its involvement in the development of the face and oral regions. The formation of the vertebrate face is in part due to the fusion of what are known as facial primordia, or facial buds and the Wnt pathway is involved in this process.

Not very much is known about the formation of the upper jaw so this study aimed to study whether the Wnt/β-catenin signaling pathway was necessary for the formation of the upper jaw in chick embryos. Previous studies had shown the Lhx8, Msx1, and Msx2 genes were all involved in the proper formation of facial and head regions of mice, and so the researchers decided to use the regulation of expression of these genes to gauge the Wnt pathway’s involvement in the formation of the upper jaw. In the previous studies when the aformentioned genes were mutated, the animal developed malformations located in the oral region (i.e. inability for proper fusion of the palatal shelf that lead to cleft palate). These genes are especially intriguing as they might be involved in cleft lip and cleft palate birth defects, which is the most common craniofacial defect found in children around the world.

The researches decided to use the chick embryo as the model organism for this experiment because the chicken embryo develops quickly and because it’s developmental conditions can be manipulated easily. The researchers also decided to use Dickkopf-1 (Dkk-1) soaked beads that were inserted to the right side of the maxillary prominence (specific region of the face) to test the Wnt pathway’s involvement; this would hopefully cause a loss-of-function of the Lhx8, Msx1, and Msx2 genes. Dkk-1 is a secreted protein that interacts with the LRP5/6 co-receptors; these co-receptors are what Wnt ligands also bind to when they bind to Frizzled to intiate Wnt pathway cascade. Dkk-1’s interactions with the LRP 5/6 co-receptors inhibit the Wnt pathway by blocking the Wnt ligangd’s ability to bind to Frizzled and the LRP 5/6 co-receptor.

From this study the researchers discovered that Dkk-1 did in fact inhibit the proper formation of the chick’s jaw area and therefore the Wnt pathway was involved. The side of the chick’s face that was treated with a Dkk-1 soaked beads had incomplete development of the bones in the upper jaw and facial region. Also when PCR’s were done for Lhx8, Msx1, and Msx2 in the presence of the Dkk-1 bead, there was definitely a decrease in expression when compared to the controls (which were left to develop normally) and the regions that were treated with Bovine Serum Albumin (which acted like the injected controls of our chick teratogenesis lab). The researchers also discovered that the Wnt pathway only affected the pattern formation of the facial structures at certain stages of development. The Dkk-1 bead only affected the formation of the upper jaw structures at an earlier stage (in this experiment it only had an affect on the HH22 stage which is about 4 days old). When the Dkk-1 soaked bead was inserted into later stage embryos the effect was either much less or there was no observable effect.

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A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer

Breast cancer is the most prevalent invasive cancer seen in women around the world. As with any cancer, breast tissue cells become cancerous when they divide in an uncontrolled manner, lose the ability to undergo apoptosis when necessary, and become undifferentiated. As such, in basil-like breast cancer, the epithelial cells that comprise the basal portion of breast tissue undergo what is called an Epithelial Mesenchymal Transition (EMT), where these cells acquire a phenotype other than the normal epithelial phenotype (e.g. dedifferentiation). This transition to a mesenchymal, stem-like phenotype is associated with metastasis to other tissues, provided these cells also possess the ability to seed a new tumor in distant tissues (not all metastatic cancer cells have this ability). Wnt signaling has been found to function in several human cancers (recall the role of the Wnt signaling pathway in liver cancer, as elucidated by Reed et al. in 2008).

Here, DiMeo et al. (2009) look at the possible role of Wnt signaling in breast cancer primary tumor formation, metastasis, and secondary tumor formation at distant sites. They investigated this in several ways. First, they compared the levels of β-catenin in primary tumor cells in mice breast tissue to that in secondary tumor cells (that arose from metastasis), noting that β-catenin levels are indicative of Wnt signaling. They show that the secondary tumor cells possessed high levels of β-catenin compared to the primary tumor cells, telling them Wnt signaling is indeed the cause of metastasis and initiation of secondary tumor formation. Second, they discovered a genetic signature present in cells of secondary tumors: genes known as slug and twist are highly expressed. These genes code for transcription factors (SLUG and TWIST), which cause the cells to undergo an EMT and metastasize to distant tissues. Finally, they examined potential inhibitors of this Wnt pathway. They discovered that Wnt inhibitor Dickkopf (DKK1) was able to inhibit this Wnt pathway by blocking the binding of LRP6 to the Frizzled receptor, disallowing Frizzled to activate Disheveled, which allows for continuing β-catenin degradation by the destruction complex (e.g. the Wnt signaling pathway is turned off). Another inhibitor, SFRP1, which binds to extracellular Wnt ligands and blocks their ability to bind to the Frizzled receptor, was unable to effectively inhibit the Wnt pathway in vivo, probably because these cancerous cells produce so much Wnt signal that SFRP1 cannot meaningfully compete with Wnt pathway activation.

The significance here is related to what we can do with this knowledge. Understanding the molecular basis of how breast cancer metastasizes and initiates tumor formation allows us to improve detection techniques, design more efficacious treatment, and save lives.

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