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Wnt signaling plays a key role in developmental processes and cell division making dysregulation to this pathway a relevant topic in many cancers. We are familiar with the frizzled receptor that binds a Wnt ligand to activate the pathway. Regulation at the frizzled receptor occurs by regular ubiqitylization. Ubiquitination works by tagging the Fzd receptor with a ubiquitin that marks the receptor for degradation by proteasomes. In this paper USP6, or deubiquitylase ubiquitin-specific protease 6 is characterized as an oncogene, a potent activator of Wnt signaling. USP6, a deubiquitylase, increases the cell surface abundance of frizzled thereby hyperactivating the Wnt pathway. Over-expression of USP6 has been observed in neoplasms, aneurysmal bone cysts, and nodular fasciitis, making the mechanism of ubiquitination important for properly targeting Wnt signaling in a subset of human cancers.
Researchers performed siRNA screens on human embryonic kidney cells (HEK293T) and human sarcoma cells (HT1080). 28 deubiquitilases were determined to be Wnt signaling regulators. The researchers concluded that silencing USP6 significantly down-regulated Wnt/β catenin reporter activity in the two cell lines. USP6 was determined to be specific to the Wnt signaling pathway but was not cell specific. Therefore, inhibition of USP6 by small molecules in different cell types may potentially benefit patients with proliferative/cancerous disorders.
Links related to the paper:
Various ubiquitin sites in the Wnt pathway.
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer death in the United States, and it consists in an adenocarcinoma (type of cancer that forms in mucus-secreting glands such as lungs). This cancer type involves mutations causing the increased activation of the EGFR tyrosine kinase pathway, and drugs have been designed to inhibit this pathway. Nonetheless, some mechanism allows these cancerous cells to survive and escape inhibition, therefore causing relapse in lung cancer. What they did in this paper was to individually inhibit all genes in the genome as EGFR inhibition was occurring as well. If the concomitant inhibition of the EGFR gene and another gene resulted in complete removal of NSCLC, then the other gene when not inhibited can be identified as a contributor to drug resistance. When the gene is normally activated in fact it allows NSCLC cells to survive and escape EGFR inhibition.
Short hairpin RNA (shRNA) screen was the technique used to knock down genes, and a specific one was used for each gene in the genome. This technique resembles the RNA interference (RNAi) we performed with C. elegans, but it works better in mammalian cells. Through this technique it was found that the Wnt pathway is involved in maintaining NSCLC cells alive during EGFR inhibition. Two poly-ADP-ribosylating enzymes were found to be particularly associated with the activation of the Wnt pathway: Tankyrase 1 and Tankyrase 2. The role of these enzymes is to positively activate the Wnt pathway by degrading Axin in the pathway, leading to survival of NSCLC cells even in the presence of EGFR inhibition.
Diagnosis of lung cancer usually occurs at a later stage when the cells have already developed mechanisms to get around the EGFR inhibition. Further understanding of how these mechanisms for survival develop might lead to new therapeutic innovations and manufacture of drugs that can efficiently decrease or eliminate NSCLC cells without any relapse events by targeting specific gene products in the pathways involved. In this case, several genes were identified as Wnt/tankyrase/fl-catenin pathway targets that can potentially be drugged to stop NSCLC cells proliferation and ultimately eliminate them.
Links related to the paper:
The telomeric PARP, tankyrases, as targets for cancer therapy - Tankyrases as Poly ADP-ribosylating inhibitors (focus on Tankyrase 1)
Role for the Related Poly(ADP-Ribose) Polymerases Tankyrase 1 and 2 at Human Telomeres - More about Tankyrase 2
Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown - Main technique description
Breast cancer is a genetically and clinically heterogeneous disease and it is not well understood how the additions of different target cells and different oncogenic mutations are connected to this heterogeneity. Evidence suggests that transgenes encode for components of the Wnt signaling pathway that potentially cause mammary cancers from progenitor cells. Expression of Wnt-1 protoonco gene in mammary glands of trangenic mice makes a population of epithelial cells expand that express progenitor cell markers, keratin 6 and stem cells antigen 1 (Sca-1). Following tumors express these markers and contain luminal epithelial and myoepithelial tumor cells that share a secondary mutation: loss of Pten which implies that this mutation arose from a common progenitor cell. Tumors arising in transgenic mice that express downstream components of the canonical Wnt signaling pathway (B-cat and c-myc) also contain a significant proportion of cells containing myoepithelial cells and cells expressing keratin-6.
Transgenic activation of different oncogenic pathways in mouse mammary glands induces tumors with different gene expression profiles, thus cells at different stages may undergo tumorigenesis as a result of the activation of different oncogenic pathways. It has been a challenge to define the specific lineage of target cells of breast cancer due to the lack of animal models expressing oncogenes in specific mammary progenitors. However, studies in the epidermis and the hematopoietic system(stem cells that give rise to other blood cells) have demonstrated that cancers that arise from stem or progenitor cells usually express markers of the originating cells. The presence of stem or progenitor cells markers in mammary tumors may suggest that the tumor arose from immature cells.
Two genes, keratin 6 and stem cell antigen 1 (Sca-1), appear to be preferentially expressed in mammary stem and/or progenitor cells. Keratin 6 is expressed in mammary gland at embryonic day 16.5 and in some of the body cells in the terminal end buds (TEBs); but keratin 6 is not found in the highly proliferative cap cells (14, 15) and rarely in cells in the mature ducts and differentiated alveoli (15), consistent with the distribution of progenitor cells. In addition, keratin 6 is associated with the arrested state of differentiation observed in the mammary ducts from C/EBP Beta-null mice. Sca-1, encoded by Ly-6A/E, is a GPI-linked protein also found in hematopoietic stem cells; it is expressed on the surface of a population of mammary cells enriched for stem cells and present in TEBs. Depletion of Sca-1-positive cells results in a loss of functional stem cells in mammary gland reconstitution (build up) experiments.
This paper reports that keratin 6 and Sca-1 cells are observed in mammary tumors induced by the Wnt-1 signaling pathway, which has been involved in proliferation and maintenance of undifferentiated cells in several tissue types, including the mammary gland (18ñ28), but not in tumors induced by oncogenes affecting other pathways. In addition, the authors provide other evidence to suggest that ectopic activation (abnormal gene expression) of the Wnt pathway may target the undifferentiated mammary progenitors for tumorigenesis.
Links related to the paper:
Initiating oncogenic event determines gene-expression patterns of human breast cancer models
Transgenic oncogene mice. Tumor phenotype predicts genotype. - Focus on 499-501
Sca-1pos Cells in the Mouse Mammary Gland Represent an Enriched Progenitor Cell Population
Apoptosis, or cell death, is essential in regulating the healthy development of organisms. Improper regulation of apoptosis can lead to many neurogenerative diseases, cancers, and other defects. In this paper, Galvin, Denning, and Horvitz study the mechanisms of apoptosis in C. elegans by screening for genes that may play a key role in regulation and singling out several main factors that affect apoptosis.
First, they performed a genetic screen to find mutations that caused an increase in programmed cell death in the P.n.aap cells of the ventral cord. They found a mutation, ced-4(n3158) that caused a cell death defect. It was discovered by another genetic screening, that extragenic mutation n3418 worked to suppress the effect of the ced-4(n3158) defect. The double mutant ced-4(n3158) n3418 causes sterility and cell reduction. They then mapped the suppressor allelic mutation (n3418) and discovered that it is a loss of function mutation of the gene spk-1. Spk-1 encodes an SR protein kinase. These proteins assist spliceosomes in regulating alternative splicing. This was particularly intriguing as ced-4 is the only known C. elegans gene with alternative splicing variants that function oppositely: ced-4L and ced-4S. Ced-4S promotes cell death whereas ced-4L prevents cell death. It was hypothesized that spk-1 plays a role in the alternative splicing of ced-4, but that has yet to be corroborated.
In summary, Galvin, Denning, and Horvitz conclude that C. elegans spk-1 inhibits apoptosis through loss of function mutation in partial loss of function alleles of ced-4. Additionally, they posit that the SR protein kinase encoded by spk-1 may assist in regulation of alternative splicing in ced-4, subsequently regulating apoptosis.
Links related to the paper:
Developmental apoptosis in C. elegans: a complex CEDnario - A review, focus on pgs 97-99
An Alternatively Spliced C. elegans ced-4 RNA Encodes a Novel Cell Death Inhibitor - Role of ced-4 gene background
Horvtiz Lab Page - Meet one of the authors who is a Nobel Prize winner!
A new method of treating cancer is to target the mitochondrial apoptotic pathway, which is regulated by the B-cell leukemia/lymphoma 2 (BCL-2) family of proteins. The apoptotic pathway is regulated by pro-apoptotic BH3-only proteins, pro-apoptotic multi-domain effecter proteins, and anti-apoptotic proteins which work together to control mitochondrial outer membrane permeabilization (MOMP), a crucial stage in completing apoptosis. The BH3 proteins are further divided into groups based on function: the activators (BID, BIM) and the sensitizers (BAD). Activators of the pathway bind to the anti-apoptotic proteins (BCL-2, BCL-XL) which results in the promotion of apoptosis.
The drug, Navitoclax (ABT-263), a small-molecule mimic of the BH3 domain of BAD, effectively binds to BCL-2 and BCL-XL, thereby releasing bound pro-apoptotic proteins and causing MOMP in BCL-2-dependent cancer cells. In early clinical trials, navitoclax showed promising results in the treatment of chronic lymphocytic leukemia (CLL) and small-cell lung cancer. However, because thrombocytes are dependent on BCL-XL for survival, a side-effect of this drug was thrombocytopenia, or abnormally low levels of platelets. From this came ABT-199, an engineered derivative of ABT-263 that maintains specificity for BCL-2 but lacks affinity for BCL-XL. ABT-199 has been shown to be potent in killing CLL in vivo in Myc-driven lymphomas in mice as well as in estrogen receptor-positive breast cancer, all while sparing platelets.
Acute myeloid leukemia (AML) cells are dependent on BCL-2 for survival, and in vitro BCL-2 inhibition by ABT-737, a compound with activity similar to navitoclax, causes cell death in AML. The goals of this study were to: (1) evaluate the anticancer effects of ABT-199 on AML and compare its effectiveness to that of ABT-737 and navitoclax - drugs that have both shown high efficacy in the ex vivo treatment of AML cells, AML primary patient samples, and in human clinical trials, and (2) determine whether BH3 profiling is a useful tool to predict response to ABT-199 treatment. BH3 profiling is a method to determine the mitochondrial priming level of a cell by exposing cellular mitochondria to standardized amounts of peptides derived from BH3 domains of BH3-only proteins and measuring the rate of MOMP, either by the timing of cytochrome c release or depolarization across the inner mitochondrial membrane. The authors used this profiling as a basis for their hypothesis that cells dependent on BCL-2 for survival will be sensitive to BCL-2 inhibition by ABT-199.
The authors research resulted in numerous findings including: that ABT-199 effectively kills AML cells in vitro and in vivo, ABT-199 sensitivity correlates with BCL-2 protein level, ABT-199-resistant MOLM-13 cells express lower BCL-2 levels compared with the parental cells, ABT-199 operates selectively on BCL-2-dependent mitochondria, ABT-199 efficiently kills primary AML myeloblasts, and ABT-199 induces apoptosis in AML stem/progenitor cells.
Links related to the paper:
The BCL-2 protein family: opposing activities that mediate cell death - Focus on pages 47 - 50
ABT-199: Taking Dead Aim at BCL-2 - Focus on page 139 to paragraph on 140 that ends with ìABT-199 causes markedly less thrombocytopenia than navitoclaxî
BCL-2 Apoptotic Pathway - Figure 2 from article 2
ABT-199 selectivity - Figure 1 from article 3
With colorectal cancers being one of the most common cancers that is diagnosed, there is a lot of interest to try and figure out ways to prevent the initiation of CRC, stop growth of CRC, or elongate the patients life that has CRC. Unfortunately, there has been only small advances in any of these efforts. The Bax protein usually helps to initiate apoptosis when it comes to mitochondrial regulation; this pathway is used in a natural way and usually benefits the organism. Bad is a part of the B-cell lymphoma 2 family. The relatives of Bax, Bcl-2 and Bcl-XL work to inhibit apoptosis, which as we know can continue cell growth and proliferation. This paper notes that when there is an increased Bcl-XL expression in colorectal cancer, there is little success in the patient's survival. This prompted them to continue more research into the effects of Bcl-XL. They used immunohistochemical staining to see if Bcl-XL was the single over expressed protein that was present in CRC. They eventually saw that Bcl-XL was a main factor in the proliferation of the colorectal tumors, which allows them to see if there is any way they can target this using drugs.
The researchers tested normal mice to see Bcl-XL's role in intestinal tissue in normal tissue and cancerous tissue. Essentially, they knocked-out Bcl-XL in these cells, which only affected a specific region (the colon), which was proven in the western blot analysis. At the end of this test, they could not find any significant data that told them there was a difference between the cell-cycle controls in the regular mice and the Bcl-XL knockout mice. They later figured that the loss of Bcl-XL would also further help mice that were tumor-induced. During the treatment the Bcl-XL knockout mice did have a slightly better status of health. Although this looked to be a positive sign, the researchers performed more tests and found some conflicting data.
The tumors that grew within the knocked out Bcl-XL mice still showed an increase of cell death. The researchers found that AOM/DSS treated mice that were also Bcl-XL knock out mice had lower tumor burden that relied only on increased cell death rate. They later tried to inhibit Bcl-XL using an inhibitor target protein. The researcher only came up with the conclusion that the data they collected was unreliable and not cohesive, which prevents them from looking into future medical uses with Bcl-XL. Although we canít see any relationship in the data among the mice, we do know that Bcl-XL is a crucial factor in the proliferation of colorectal cancer because it protects the cell from death.
Links related to the paper:
Basic Background on Colorectal Cancer:
The Relationship between Bax and the Bcl-2 family (in relation to diabetes) - Although this is related to diabetes, the Bax and Bcl-2 proteins have the same relationship in colorectal cancer.
More information on Bcl-XL and Bcl-2 and what they do:
http://onlinelibrary.wiley.com/doi/10.1111/j.1582-4934.2003.tb00225.x/epdf
Increased Bcl-XL expression shown to inhibit senescence and apoptosis in pancreatic neoplasia
This paper examines the roles of the RTK pathway in hair growth and development. Specifically, it examines the roles of RAS and FGFR and their effects on downstream pathways, including the regulation of Shh, a known factor in hair development.
Normal hair growth is characterized by a number of factors, including length, distribution, density, and number of bends and medulla. These are effected by RAS mutations, which are present in a number of cancers and genetic disorders related to developmental abnormalities, often effecting length, dispersion, and shape of hair. When mutant Kras, which is found to be over-active in cancers and genetic disorders, is expressed in the skin it results in hair loss. To examine the results of mutant Kras in hair follicles, the researchers used a LSL-Kras model in which the expression of mutant Kras was regulated by the Msx2 allele, which is expressed in the matrix of the postnatal hair follicle, the dorsal skin during embryonic development, and the apical ectodermal ridge of the early limb bud.
The Msx2-Cre;LSL-KRASG12D transgenic mice were viable without changes in the limb. By 1-2 weeks they exhibited abnormal hair growth, with changes in shape and reduction in length. They found the reduction in hair length to be a result of decreased proliferation and growth rate. To determine the context in which these changes occurred, they examined and found significantly altered expression of many genes in downstream pathways. They found a significant down-regulation of Shh and itís targeted genes, suggesting the increased signaling in the RTK pathway resulted in decreased Shh ligand expression and reduced Hedgehog pathway signaling.
In particular, they found regulation of Shh RNA by FGF2 to be the most altered. To examine this further, they used a mutant Fgfr2 model. These mice developed normal skin and hair until 2 weeks, after which it became long, shiny and brittle. In these mice, Shh mRNA was 2-fold higher than in controls, corroborating the theory that active FGF2 downregulates Shh. By comparing the effect of EGFR2 to a known inhibitor of transcription, they determined that FGF2 works to repress Shh by blocking transcription. Based on these results, the researchers purport that KRAS normally functions to activate Fgf2, which suppresses Shh.
Links related to the paper:
Epidermal Patterning and Induction of Different Hair Types during Mouse Embryonic Development - Paper describing normal hair development in mice
Cre-Lox System - Image explaining breeding of Cre-Lox mice
Cre-Lox System - Image explaining the Cre-Lox system
Sonic hedgehog signaling is essential for hair development
The otic placode is responsible for creating the inner ear that is responsible for hearing and balance. The otic placode in the early embryo is similar to the lens placode where a section of ectoderm thickens and forms into an otic cup then into a vesicle that contains the cochleovestibular ganglion that help relay sound vibrations to the brain for processing. In mice, previous experiments have shown that otic vesicles can be reduced in size when Wnt signaling is inhibited in that area and conversely they can be increased in size through ectopic activation of Wnt signaling. Also Fibroblast Growth Factor (FGF) dependent RTK signaling can induce larger otic vesicles when over-expressed or inhibit otic tissue differentiation when inhibited. Therefore, both of these types of cell-cell signaling are tightly regulated in inner ear development, but we don't know exactly how.
Wright et al wanted to investigate how Wnt and RTK signaling interact and if they in fact cooperate in early inner ear development in the mouse. Previously it has been seen that in the vertebrate model organisms: Xenopus, chick, and zebrafish the Wnt and RTK signaling cooperate during the induction of the otic placode tissue but it is unclear in mice. They studied the spry1 and spry2 FGF antagonizing gene expression for indication of an active RTK pathway and also Wnt reporter fl-catenin expression for an indication of an active Wnt pathway. In another experiment they created spry1, spry2, and mutants fl-catenin by floxing these target genes to determine what happened to otic placode formation in the presence or absence of effective RTK or Wnt signaling.
They saw the expression of RTK signaling earlier on and in a larger region than Wnt signaling. They also saw that in mutants where RTK signaling was inhibited that Wnt activity increased. Through these in vivo embryological experiments they found that RTK signaling is actually responsible for activating Wnt signaling in mouse early inner ear development, therefore they cooperate.
Links related to the paper:
Helpful Review Article on Otic Placode Formation in Vertebrates (Chick):
Article about Otic Placode Induction in Mouse see Figures for nice Otic Placode Cross Sections:
In the 2015 study conducted by Chein et al., the researchers wanted to determine the role of a Wnt signaling pathway in the polarization of the anterior lateral microtubule, or ALM, neuron in C. elegans. ALMs are typically found as a pair of neurons with the body of the neuron located at the posterior end of the worm. A process extends from the body of the ALM toward the anterior end of the worm, and function as touch sensors. It is known that the polarization of PLM neurons, which are related to ALM neurons, is controlled by the signal from Wnt LIN-44 to the Frizzled receptor LIN-17.
The study used RNAi bacteria fed worms to knock out certain genes that encode proteins that function in a non-canonical Wnt signaling pathway. The knock out of single genes, such as cam-1 and mom-5, caused defects in ALM polarization. It was found that CAM-1 and MOM-5 act as co-receptors and regulate Wnt signaling. Overall, the study showed that CAM-1 has a duel function in Wnt signaling regulation, which in turn controls ALM polarity. CAM-1 can either function as a receptor to promote Wnt signaling in the ALM, or act as an inhibitor to stop Wnt signaling outside the ALM.
Links related to the paper:
Neuron Body Plan Information - Look mainly at processes
RNAi Information - Look specifically at RNAi by feeding
Information on co-receptors - Box1 should be sufficient for basic understanding of co-receptors
Information on Wnt Signaling - Focus mainly on non-canonical Wnt signaling and HSN migration
