1,720,961 research outputs found
Lamellipodia as Proliferation Machines: How Branched Actin Based Feedback Loops Allow Melanoma to Evade Growth Inhibition
No full textThe general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Spatial regulation of Rac1 activity is well-characterized and important for several cellular functions, including building and sustaining protrusive branched actin networks at the cell edge. High Rac1 activity sits atop a dense cytoskeletal scaffold that drives the extension of a flat lamellipodium, whose tightly packed volume forms a microdomain of enhanced signaling activity with elevated transduction efficiency. Cancer cells can coopt this mechanism to drive proliferation and survival signaling. For example, the hyperactive Rac1P29S mutation found in 10% of melanoma patients is associated with advanced disease, increased resistance to MAPK pathway inhibitors, and decreased patient survival. Previous work demonstrated that these clinical phenotypes correlate at a cellular level with sustained proliferation under drug challenge and is linked to a cell's ability to build extended lamellipodia. Using FRET- and localization-based biosensors for Rac1 activity, we observe larger and more potent microdomains in melanoma cells expressing Rac1P29S. The resultant branched actin network provides abundant binding sites for the tumor suppressor NF2/merlin. Superimposition of heightened Rac1 activity poises the Rac1 effector kinase PAK to inactivate merlin through phosphorylation. Displacement of merlin from the lamellipodia by a competitive actin-binding peptide interrupts this phospho-inactivation, re- sensitizing melanoma cells to MAPK inhibition. However, merlin plays an additional role upstream of the Rac1 signaling domain. Merlin recruitment to the cell edge limits protrusion formation. Knockout of merlin recapitulates the Rac1P29S-induced phenotype, producing enhanced lamellipodia with elevated wildtype Rac1 activity upon MAPK-inhibition. Breaking merlin's inhibition of the branched actin network similarly allows for enhanced lamellipodia formation, and ultimately merlin phospho-inactivation. Together these data support a double-negative feedback--Rac1 microdomains inactivate merlin localized to the dense branched-actin networks; active merlin, in turn, attenuates microdomain formation through inhibition of branched actin polymerization. This localized signaling feedback is thus prone, when perturbed, to produce self-sustaining patterns of signaling activity, allowing for MAPK-inhibited melanoma to escape merlin-mediated growth control
Glutamine Antagonism and Its Utility as a Therapeutic Modality in Cancer
Get PDFGlutamine metabolism is important in cancer as it fuels the TCA cycle, plays a role in redox homeostasis, and contributes to the production of nucleotides, amino acids, and lipids for survival, making glutamine metabolism a promising target in cancer therapy. The work outlined in this dissertation focuses on understanding the mechanism of the broad glutamine metabolism inhibitor, 6-diazo-5-oxo-L-norleucine (DON) and its prodrug, JHU-083, while comparing them to the effects of CB-839, a specific glutaminase inhibitor. DON is one of the oldest and well-known glutamine antagonists and can effectively limit tumor growth in a preclinical setting. Unfortunately, DON was removed from early phase clinical trials due to unacceptable toxicity in the gastrointestinal tract (GI). Thus, DON prodrugs were recently developed to be inactive until cleaved by cathepsins enriched in the tumors or by plasma esterases, bypassing toxicity in the GI tract.
Using isotope tracer studies in cancer cells and mouse xenograft models, I found that DON and JHU-083 mainly inhibit glutamine-derived nitrogen labeling in purines but unexpectedly does not limit the contribution of glutamine-derived carbon labeling of tricarboxylic acid (TCA) cycle metabolites. Additionally, I found that DON and JHU-083 can limit the levels of purines but not the levels of most TCA cycle metabolites. These findings suggest that these drugs are poor inhibitors of glutaminase in the cancer cell lines tested and that DON and JHU-083 mainly target purine metabolism.
Recognizing DON and JHU-083 as effective purine metabolism inhibitors can offer insight into which cancer patients could benefit from these drugs. Relapsed small-cell lung cancer (SCLC) is characterized by an upregulation of de novo purine biosynthesis and have few durable therapies. Using metabolic tracing and untargeted metabolomics, I found that DON can inhibit purine metabolism in treatment-naïve and chemoresistant pairs of SCLC. In a mouse xenograft model of relapsed SCLC, JHU-083 induces a delay in tumor growth without overt side effects. My work provides an opportunity to explore JHU-083 as an anti-cancer therapy for diseases that depend on purine biosynthesis
Decellularized Normal and Cancer Susceptible Mice Colons to Study the Contribution of the Extracellular Matrix to Cell Behavior and Colon Cancer Progression
Get PDFCurrent 3D culture models to study colorectal cancer lack architectural support and signaling proteins provided by the tissue extracellular matrix (ECM) which may influence cell behavior and cancer progression. Therefore, the ability to study cancer cells in the context of a matrix that is physiologically more relevant and to understand how the ECM affects cancer progression has been understudied. To address this, I developed an ex-vivo 3D system, provided by intact wild type (WT) and colon cancer susceptible decellularized mouse colons (DMC), to support the growth of human cancer cells. DMC are free of viable cells but still contain extracellular matrix proteins including subsets of collagens. Stiffness, an important mechanical property, is also maintained in DMCs. Importantly, I observed that the DMC is permissive for cell proliferation and differentiation of a human colon cancer cell line (HT-29). Notably, the ability of cells in the WT DMC to differentiate was also greater when compared to Matrigel TM, an extracellular matrix extract from a mouse tumor cell line. Additionally, I observed using invasion assays that DMC obtained from polyps from a colon cancer susceptible mouse model facilitated increased cell migration/invasion of colorectal cancer cells and immortalized non-tumor colonic epithelial cells compared to DMC from WT mice. Finally, using mass spectrometry, I identified extracellular matrix proteins that are more abundant in DMC from a colorectal cancer mouse model compared to age and sex-matched WT mice. I propose that these abundantly expressed proteins in the tumor microenvironment are potentially involved in colorectal cancer progression
Pleiotrophin and Midkine Confer Metastatic Ability to Disseminated Breast Cancer Cells
No full textMetastasis is a highly inefficient process with several rate limiting steps. It is estimated that only 0.01% of disseminating tumor cells are successful in forming overt metastasis. To understand the biology behind successful metastasis formation, we performed differential transcriptomic analyses between a pair of highly metastatic and poorly metastatic syngeneic mouse breast cancer lines. We found Pleiotrophin (PTN) to be enriched in metastatic breast cancer in murine and human patient samples. By developing tools to visualize the tumor microenvironment, we found that PTN produced by metastasis initiating cells in secondary organs can create an immune suppressive niche made up of neutrophils that induce T cell dysfunction. Consequently, blocking PTN function reversed local immune suppression and sensitized triple negative metastatic breast cancer to immune checkpoint blockade and chemotherapy. Encouraged by the promising results from PTN blockade, we studied the function of its only other family member Midkine (MDK) in breast cancer progression. We found MDK expression to correlate with poor prognosis in breast cancer patients. Unlike PTN, MDK was widely expressed in primary and secondary tumors suggesting a more diverse function of MDK. Blocking MDK in vivo led to a significant decrease in metastasis to lungs. Given their functional importance in metastatic disease, further investigation into the functional overlap and differences between MDK and PTN is warranted. Understanding their unique biology will lead to development of novel therapeutic strategies that break the immune suppression of immunological hot cancers such as triple negative breast cancers
Analysis of Meiotic Cancer Testis Antigens and Their Contribution to Tumorigenesis
Get PDFTumors frequently activate the expression of genes that are only otherwise required for meiosis. These Cancer Testes Antigens (CTAs) have been found to play critical roles in multiple cellular processes when aberrantly expressed in cancer. Here I found that the CTAs SPO11, GAGE and HORMAD1 play critical roles in cell survival, modulating response to DNA damaging agents, and DNA replication stress mitigation respectively. More specifically, we find HORMAD1 is critical for protecting stalled DNA replication forks in LUAD. Loss of HORMAD1 leads to nascent DNA degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. Moreover, following exogenous induction of DNA replication stress, HORMAD1 deleted cells accumulate single stranded DNA (ssDNA). We find that these phenotypes are the result of a lack of RAD51 and BRCA2 loading onto stalled replication forks. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal aberrations in response to replication stress. Collectively, our work supports the hypothesis that CTAs become abnormally expressed in cancer to buttress oncogenic behaviors and given their restrictive expression to the testes and tumors, they are unique targets with a broad therapeutic window
The Role of Protocadherin 7 in Lung Adenocarcinoma
Get PDFLung cancer is the leading cause of cancer-associated deaths worldwide. Despite recent advances in the development of targeted therapies for lung cancer, most patients develop resistance to these targeted therapies. Our lab has uncovered a key oncogenic role for Protocadherin 7 (PCDH7) in non-small cell lung cancer (NSCLC) (Zhou et al. Cancer Research, 2017; Zhou et al. Molecular Cancer Research, 2019). PCDH7 is frequently overexpressed in human lung cancers, significantly correlating with poor clinical outcome of lung adenocarcinoma patients. PCDH7 knockout in established lung tumor xenografts led to a significant decrease in phospho-EGFR, leading us to hypothesize that PCDH7 may directly interact with EGFR and modulate signaling through this receptor. We demonstrate that PCDH7 interacts with EGFR in a phospho-dependent manner, and that loss of this interaction in PCDH7 truncation mutants reduces EGFR activity and downstream signaling. PCDH7 knockout cells also exhibit decreased EGFR dimerization, suggesting a model whereby PCDH7 promotes EGFR dimerization to stimulate downstream MAPK activation. We also investigated the consequences of PCDH7 loss of function in EGFR mutant human cells and mouse models. Knockout of PCDH7 sensitizes EGFR mutant cell lines to tyrosine kinase inhibitors (TKIs), the current method of treatment for EGFR mutant lung cancers. Furthermore, loss of PCDH7 in EGFR mutant xenografts and genetically engineered mice reduces overall tumor burden. Overall, these findings reveal a new mechanism through which PCDH7 potentiates the MAPK pathway and provide strong rationale for the development of PCDH7-targeting molecules including monoclonal antibodies
Delineating Mechanisms of Signal-Induced RNA Polymerase II-Transcription
No full textSignal-induced transcriptional programs regulate critical biological processes through the precise spatio-temporal activation of inducible gene programs. Understanding the dynamics by which RNA Polymerase II precisely induces transcription to activate these programs is important for dissecting the basis for their role in cell fate responses and disease progression. Here, we utilize high-resolution genomic approaches coupled with temporal signal induction to characterize how individual transcription steps contribute to the gene expression cycle in signal-induced transcription activation. Our first story (chapter 2) utilizes acute depletion approaches to reveal that the KAP1 protein is a positive regulator of transcription of immediate early genes, a class of signal-induced genes that regulate diverse biological processes including cancer and development. Mechanistically, KAP1 negatively regulates elongation rate at the early stages of transcription, which allows for proper kinetic progression through the transcription cycle by bolstering new initiation and full activation of gene expression. Overall, this study is the first report to link KAP1 "repressive" control of transcription to a positive role in gene activation and has implications for transcription-induced cell fate responses. Our second story (chapter 3) centers on ligand-induced transcription activation of the HIV-1 provirus. Decades of research has shown that HIV-1 transcription is primarily activated through pause release and transcription elongation by the HIV-1 transactivator Tat. Here, we use a novel Tat depletion approach to show that Tat function in pause release is the catalyst that promotes sustained RNA Polymerase II recruitment to the HIV-1 promoter. This recruitment of RNA Polymerase II is the mechanism that robustly induces "logarithmic" HIV-1 expression and viral replication. These data reveals a new significance for Tat function in transcription initiation and explains how Tat can sustain extended levels of HIV-1 transcription for proviral fate. Overall, the two studies have provided important mechanistic insights for understanding transcription dynamics in gene expression programs that have implications for diverse biological phenomena and pathogenesis
Novel Functions of the Transcription Factor Aryl Hydrocarbon Receptor (AHR) and Its Tryptophan-Derived Ligands in Cancer Cells
Get PDFThis comprehensive study delves into the metabolic reprogramming of cancer cells, focusing on the proto-oncogene MYC and the aryl hydrocarbon receptor (AHR). The role of MYC in regulating tryptophan (Trp) metabolism was investigated in colon and liver cancer cells using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our findings reveal that MYC enhances the intracellular levels of Trp and its metabolites in the kynurenine pathway, specifically increasing the expression of Trp transporters SLC7A5, SLC1A5, and the enzyme AFMID. Elevated levels of these components were observed in immortalized colon cancer cells lines and patient tissues, with a significant increase in kynurenine. Blocking enzymes in this pathway led to the preferential death of cancer cells, suggesting a potential therapeutic strategy. Furthermore, our research highlights the role of AHR in cellular detoxification and proliferation, particularly in MYC-overexpressing cells. We found that AHR knockdown reduced the expression of genes crucial for metabolic pathways necessary for cell proliferation, such as LDHA, DHODH, and UMPS. Additionally, we identified SCIN, an actin-severing protein, as a key target of AHR in colon cancer cells, necessary for cell proliferation and activation of the WNT pathway through β-catenin. In liver cancer, we discovered that MYC-driven tumors have a critical dependence on Trp, with a diminished utilization of the Trp via the Kyn pathway. Depriving these tumors of Trp inhibits their growth, while supplementation with the Trp metabolite I3P restores growth, presenting a novel therapeutic target. Overall, our findings underscore the importance of MYC and AHR in regulating amino acid metabolism in cancer and open new avenues for targeted cancer therapies
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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