Category C. Cancer Biology / Molecular Biology
C1. Genetic control of tissue specific growth in the larval trachea of Drosophila
Paulo Leal, Cyril Cook, Brett Bohmann, Alex Matlock, and Robert Ward
Department of Molecular Biosciences, University of Kansas, Lawrence, KS
Different tissues and organs grow at different rates compared to overall organismal growth, suggesting growth mechanisms that act tissue specifically. The mechanisms of tissue specific growth are less well understood than those governing the growth of an entire organism. To gain a better understanding of tissue specific growth processes we have been characterizing mutations that alter growth in single tissues in genetically tractable model systems. Larval trachea growth in Drosophila melanogaster is well suited for these studies since the trachea shows dramatic growth during the larval stages, can be imaged and measured in living animals, and gene expression can be specifically altered in this tissue using breathless-GAL4. In addition mutations in uninflatable (uif) and Matrix metalloproteinase 1 (Mmp1) are larval lethal with phenotypes including specific defects in larval tracheal growth relative to overall organismal growth. Here we report the characterization of 8 additional tracheal growth mutations recovered from screens of an EMS mutagenesis and larval lethal P elements. We are conducting phenotypic analyses of the mutant animals coupled with whole genome sequencing to identity the causative mutation in each line. Since the growth of larval endoreplicating tissues (including the trachea) is fueled by nutrition and largely regulated through the insulin signaling pathway, it is possible that mechanisms of tissue specific growth in this tissue will impinge upon insulin signaling. Therefore we are analyzing the insulin signaling pathway in these mutants and attempting to rescue their growth defects by upregulating insulin signaling specifically in the trachea.
C2. An examination of septate junction components in morphogenesis of Drosophila melanogaster
Sonia Hall, Beth Jarvis and Robert E. Ward IV
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045
Organismal development depends upon highly regulated cellular rearrangements and cell shape changes. These morphological processes require coordination between signaling pathways that regulate actin dynamics to propagate forces through tissues and stabilization of these effects mediated by adherens junctions. Our lab conducted a screen for genes that regulate leg morphogenesis during metamorphosis in Drosophila. From this screen we identified Macroglobulin complement related (Mcr). During the characterization of Mcr, we found that it serves an essential role in the establishment of septate junctions (SJs). SJs, similar to vertebrate tight junctions, are occluding junctions along the lateral membranes of epithelial cells that allows for the separation of chemically distinct organ compartments. Previous work indicated that a few SJ genes play a role in morphogenesis including Coracle, NeurexinIV, and Discs Large in dorsal closure, and Gliotactin and Coracle in imaginal disc morphogenesis. To determine whether the SJ as a complex functions in morphogenesis, or if only a few individual SJ genes have additional roles in regulating morphogenesis, we conducted an analysis of twelve SJ mutations in coordinating complex morphogenetic processes during embryonic, larval, and pupal development. From this analysis, we conclude that each SJ component examined has an essential role in regulating morphogenesis during development. We propose the SJ complex has a novel, essential role in morphogenesis prior to its assembly into the occluding junction. Next, we will examine how SJ components function as a complex or sub-complex to regulate morphogenesis.
C3. Anti-cancer agent (-)-gossypol inhibits colon cancer cells by targeting RNA-binding protein Musashi1
Lan Lan1, Carl Appelman1, Amber Smith1, Sarah Larsen1, Jia Yu1, Rebecca Marquez1, Hao Liu1, Philip Gao2, Na Zhang2, Anuradha Roy3, Asokan Anbanandam4, Ragul Gowthaman1,5, John Karanicolas1,5, Jeffrey Aubé6, Kristi Neufeld1, and Liang Xu1
1Department of Molecular Biosciences, 2Protein Production Group, NIH COBRE in Protein Structure and Function, 3High Throughput Screening Laboratory, 4Bio-NMR Core Facility, 5Center for Bioinformatics, 6Department of Medicinal Chemistry, The University of Kansas.
Musashi1 (Msi1) is an RNA-binding protein that acts as a translational repressor by binding to Msi1 consensus binding sequences (MCSs) within the 3'-UTRs of target RNAs and inhibiting their translation. The best characterized RNA target of Msi1 is Numb, which encodes a protein that inhibits Notch signaling. Other known RNA targets of Msi1 translational inhibition include APC and P21WAF-1, which encode inhibitors of Wnt and p53/Rb signaling, respectively. Given that many cancers exhibit increased Notch, Wnt, and/or p53/Rb signaling, we reasoned that increased expression of Numb, APC and P21WAF-1 through inhibition of Msi1’s RNA binding activity might be an effective way to simultaneously decrease Notch, Wnt and p53/RB signaling activity and thus slow the growth of a broad range of cancers. With this goal in mind, we assayed the ability of ~55,000 small molecules to disrupt the binding of Msi1 protein to its consensus RNA binding site in numb mRNA. One of the top hits was (-)-gossypol ((-)-G), a natural compound from cottonseed, with previously shown potent anti-tumor activity and completed phase IIb clinical trials. Here, we use surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) studies to demonstrate a direct interaction of (-)-gossypol with the RNA binding pocket of Msi1. We further show that (-)-gossypol reduces Notch and Wnt signaling in a variety of colon cancer cell lines. We also show that (-)-gossypol reduces survivin expression and increases apoptosis. Finally, we show that (-)-gossypol inhibits colon cancer cell proliferation in a mouse xenograft model. Our data significantly add to a growing body of evidence that inhibition of Msi1 is an effective anti-cancer strategy, and identify (-)-gossypol as a promising lead compound in the development of Msi1 inhibitors.
C4. Identification of drugs that specifically deplete mutant p53 in cancer
Alejandro Parrales1, Scott Weir2, Anuradha Roy3, and Tomoo Iwakuma1
1 Department of Cancer Biology, University of Kansas Medical Center, Kansas City, Kansas, USA.
2 Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas, USA.
3 High Throughput Screening Laboratory, Del Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas, USA.
Mutations in the tumor suppressor p53 are detected in more than 50% of human cancers and are associated with genomic instability, metastasis, chemotherapeutic resistance, higher clinicopathological grading, and poor patient’s prognosis. Most p53 mutants not only lose the transcriptional activity (loss of function), but also gain oncogenic properties (gain of function), such as metastatic potential and resistance to chemotherapeutic drugs. We and others have recently demonstrated that depletion of p53 mutants in cancer cells significantly reduces their malignant properties, providing the rationale to target mutant p53 as an anti-cancer therapeutic. A drug that depletes mutant p53 but not wild-type p53 would affect only cancer cells with little effect on normal tissues, since mutations in the p53 gene are detected mostly in cancer cells alone. In order to identify chemical compounds that specifically reduced mutant p53 expression, we performed high-throughput screens for 9,718 chemical compounds using an osteosarcoma cell line Saos2 expressing a hotspot mutant (p53R175H) fused with luciferase reporter (Luc-p53R175H); Saos2 cell line expressing luciferase alone was used as a negative control (Luc). Eighty-six (86) compounds showed a dose-dependent inhibition of luciferase activities in Luc-p53R175H cells; however the majority of them inhibited the luciferase activity of Luc cells as well. We selected several compounds showing significant difference in the inhibition of luciferase activities between Luc-p53R175H and Luc cells. However, many compounds showed cytotoxicity, no reduction in mutant p53 levels, or reduction in both wild-type and mutant p53 levels. Finally, we identified two structurally related compounds that successfully reduced the levels of several p53 mutants with little effects on wild-type p53 level. These compounds did not alter RNA expression of the p53 gene. Interestingly, they reactivated wild-type p53 transcriptional activity in cancers cells harboring mutant p53 alone and transactivated RNA expression of several p53 target genes. Furthermore, their effects on mutant p53 were partially rescued by Nutlin3a, a drug to interfere the MDM2-p53 interaction, suggesting that MDM2 was involved in the mutant p53 depletion by these compounds. Together, our results propose that these compounds alter the protein conformation of mutant p53 and induce the degradation by its ubiquitin ligase MDM2. Our identified compounds can be used for cancer therapy via attenuation of oncogenic gain-of-function activity of mutant p53, which will significantly improve current therapeutic modalities and potentially extend survival in many cancer patients.
C5. Lysyl Oxidase-like 2 Secreted from Breast Cancer Cells Undergoes Proteolytic Activation
Trey Ronnebaum, HeeJung Moon, Joel Finney, Mason Lantz, Minae Mure*
Department of Chemistry, Mure Lab Research Group, University of Kansas, Lawrence, KS
Human lysyl oxidase-like 2 (LOXL2) has been considered as a promising therapeutic target since it is highly up-regulated in metastatic breast cancer cells and tissues. Expression of LOXL2 also induces epithelial-to-mesenchymal transition (EMT, the first step of metastasis/invasion) promoting cell proliferation and invasion. Inhibiting the production and activity of LOXL2 by shRNA and a specific antibody has shown to retard the progression of breast cancer in cells and in mice models. However, the molecular mechanisms whereby LOXL2 functions in breast cancer metastasis/invasion still remain elusive. This is mostly due to the lack of recombinant LOXL2 (rLOXL2) suitable to conduct functional studies of LOXL2. We have recently hurdled this obstacle and produced >95% pure and soluble rLOXL2 differing the extent of PTMs, subcellular localization and catalytic competencies. In this study, we will describe that N-glycosylation at Asn455 and Asn644 of LOXL2 is essential for protein secretion and that secreted LOXL2 undergoes proteolytic activation in the ECM.
C6. Metastasis suppressor MTBP inhibits the Erk1/2-Elk-1 signaling pathway in hepatocellular carcinoma
Atul Ranjan1, Qian Bi1, 2, Rui Fan2, Neeraj Agarwal1, Danny R. Welch1, Steven A. Weinman3, Jie Ding2, Tomoo Iwakuma1
1Department of Cancer Biology, 3 Pharmacology, University of Kansas Medical Center, Kansas City, KS 66010, USA, 2State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, P.R. China
MDM2 Binding Protein (MTBP) is implicated in the progression of several types of cancer as a metastasis suppressor. However, it remains unsolved how MTBP contributes towards cancer progression and which signaling pathways MTBP regulates. Our previous studies indicate that MTBP suppresses cancer metastasis by regulating cell migration. MTBP binds to a-actinin 4 (ACTN4) and inhibits its actin-crosslinking function, resulting in the suppression of ACTN4-mediated filopodia formation and cell migration. In this study, we found that reduced MTBP expression in human hepatocellular carcinoma (HCC) tissues was correlated with capsular/vascular invasion and lymph node metastasis. We also found that MTBP suppressed HCC cell migration via pathways other than ACTN4. In order to identify signaling pathways altered by MTBP, we used luciferase-based signal arrays and found that MTBP overexpression inhibited the transcriptional activity of Elk-1, an Ets-oncogene family transcription factor, to 60% of the control. MTBP also inhibited phosphorylation of Elk-1, as well as expression of Elk-1 target genes, c-fos, EGR-1, and PKCa, upon EGF stimulation. Elk-1 is phosphorylated by nuclear phosphorylated Erk1/2 (p-Erk) and activated as a transcription factor. We therefore examined the effects of MTBP overexpression on the phosphorylation status and cellular localization of Erk1/2. Indeed, MTBP inhibited the nuclear translocation of p-Erk. Since p-Erk is mainly imported to the nucleus by Importin7/RanBP7, we furthermore investigated functional correlation between MTBP and Importin7/RanBP7. We successfully demonstrated that MTBP endogenously interacted with Importin7/RANBP7 and inhibited the interactions between p-Erk1/2 and Importin7. These results demonstrate a novel function of metastasis suppressor MTBP, suggesting that MTBP suppresses HCC metastasis by regulating the Erk1/2-Elk-1 pathway which plays a crucial role in migration and metastasis of many cancer types.
C7. Nuclear Adenomatous polyposis coli (Apc) suppresses colitis-associated tumorigenesis in mice
Maged Zeineldin1, Matthew A. Miller1, Ruth Sullivan2 and Kristi L. Neufeld1
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS. 2University of Wisconsin, Madison, Carbone Cancer Center and Research Animal Resources Center
Mutation of tumor suppressor APC initiates most human colorectal cancers and chronic colitis can significantly increase risk. APC is a nucleo-cytoplasmic shuttling protein with numerous proposed functions. We recently generated a mouse model with mutations that inactivate the Apc nuclear localization signals (ApcmNLS) to examine roles for nuclear Apc in the context of a whole organism. We now report that ApcmNLS/mNLS mice have increased susceptibility to tumorigenesis induced with Azoxymethane (AOM) and Dextran Sodium Sulfate (DSS). The AOM-DSS induced colon adenoma histopathology, proliferation and b-catenin and Kras mutation spectrums were similar in both ApcmNLS/mNLS and Apc+/+ mice. However, AOM-DSS-treated ApcmNLS/mNLS mice showed more weight loss and more lymphoid follicles than Apc+/+ mice, hinting of a predisposition to colitis. To test this hypothesis, we induced acute colitis with a 7 day DSS treatment followed by 5 days of recovery. Compared to Apc+/+ mice, DSS-treated ApcmNLS/mNLS mice developed more severe colitis based on clinical grade and histopathology. ApcmNLS/mNLS mice also had higher lymphocytic infiltration and reduced expression of stem cell markers, suggesting an increased propensity for chronic inflammation. Moreover, colons from DSS-treated ApcmNLS/mNLS mice showed goblet cell defects and reduced Muc2 expression. Even in untreated ApcmNLS/mNLS mice, there were significantly fewer goblet cells in jejuna, and a modest decrease in colonocyte Muc2 expression compared to untreated Apc+/+ mice. Colonocytes from untreated ApcmNLS/mNLS mice also showed increased expression of inflammatory mediators; cyclooxygenase-2 (Cox-2) and macrophage-inflammatory-protein-2 (MIP-2). These findings reveal novel functions for nuclear Apc in goblet cell differentiation and protection against inflammation-induced colon tumorigenesis.
C8. TGF-β decreases APC protein by inducing Musashi-1 expression in intestinal cells
Andy R Wolfe1, Fernando F. Blanco2, Erick Spears1, Dan A. Dixon2, Kristi L Neufeld1,2
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS
2Department of Cancer Biology, University of Kansas Medical School, Kansas City, KS
RNA binding proteins play critical roles in post-transcriptional gene regulation controlling many different events including mRNA stability, transport, and translation. Musashi 1 is an RNA binding protein that has two domains that bind to the 3’ UTR of target mRNA and block translation. The most characterized target of Musashi 1 is Numb, an antagonist of the Notch pathway. Other identified targets support a role for Musashi 1 in apoptosis, differentiation, proliferation, and cell cycle. The intestine is lined with a layer of epithelial cells that contains invaginations called crypts and is continuously replenished. Stem cells at the bottom of the crypt give rise to transient amplifying cells which continue to proliferate and then eventually differentiate as they reach the top of the crypt at which point they die and are sloughed in to the lumen. Within the intestine, Musashi 1 is expressed in the lower portion of the crypt as well as throughout the transient amplifying region. One target mRNA of Musashi 1 we have identified is the tumor suppressor adenomatous polyposis coli (APC). The most characterized function of APC is in the Wnt signaling pathway where APC participates in destruction of oncoprotein β-catenin. The Wnt pathway plays an important role within the intestine to control proliferation of the epithelial cells within the lower portion of the crypt. We have already demonstrated that Musashi 1 blocks translation of APC mRNA and current data shows that over expression of Musashi 1 stabilizes APC mRNA in cell culture. Another signaling pathway important for homeostasis of the intestine is the TGF-β pathway which typically blocks proliferation of cells and is activated as the cells near the top of the crypt. The coordination of these pathways is critical for normal development. We previously reported that untransformed lung epithelial cells (Mv1Lu) treated with TGF-β show a decrease in APC protein but not mRNA. The underlying mechanism for this unexpected observation was not clear. Here we show a similar phenotype in intestinal epithelial cells and we provide evidence that APC mRNA levels are stabilized by Musashi 1 expression which is induced by TGF-β.
C9. Adenosine A3 receptor signaling inhibits stem-like properties of osteosarcoma
Swathi V. Iyer, Neeraj Agarwal, Alejandro Parrales, and Tomoo Iwakuma
Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS, USA.
Osteosarcoma (OS), the most common type of bone cancer, is the second leading cause of cancer-related death in children and young adults. Despite advances in the treatment for OS, the survival rate of high-grade OS has reached a plateau remaining at 50-80% for the past three decades mainly due to its high metastatic and drug-resistant properties. Increasing evidence indicates that only a small population of cells within the tumor has high abilities to self-renew and reinitiates tumors comprising of a diverse cellular population. This highly malignant cellular population possessing stem-like properties is considered to be responsible for tumor progression and recurrence, and hence referred to as tumor initiating cells (TICs) or cancer stem cells (CSCs). The inadequacy of current treatments may be a result of our inability to target TICs in OS. We previously illustrated that as few as 200 cells from OS spheres that grow in anchorage- and serum-independent conditions efficiently give rise to tumors in immunocompromised mice and possess high proportion of cells expressing stem cell-associated markers. Thus, sphere forming potential is well correlated with malignancy and stem-like properties of cancer cells. To identify factors that regulate sphere formation of OS cells, we performed screenings of a human whole genome shRNA lentiviral library in SJSA-1 OS cell line possessing a baseline of sphere-forming ability. These screenings successfully identified adenosine A3 receptor (A3AR) whose downregulation significantly increased sphere formation. We therefore hypothesized that A3AR is crucial in the suppression of OS malignancy by regulating the stem-like properties. Our molecular and biological analyses revealed that A3AR downregulation significantly increased migratory potential of SJSA-1 cells, as well as their abilities to form tumors and metastasize. We also found that A3AR knockdown enhanced stem-like properties, such as increase in the expression of stem cell marker Oct-4, aldehyde dehydrogenase (ALDH) activity, ability to differentiate into the adipogenic lineage, and tumor initiating potential. We furthermore demonstrated that A3AR downregulation increased nuclear NF-κB and β-catenin levels, both of which play key roles in OS malignancy. Finally, treatments of aggressive OS cell lines with an A3AR agonist CF101 significantly reduced their sphere forming potential. Thus, our data strongly suggest that A3AR signaling plays a crucial role in malignancy and stem-like properties of OS. Taken together, our study has discovered A3AR as a novel regulator of the malignant nature of OS, making it an attractive and efficient therapeutic intervention for high grade OS.