Student News and Views
KLF5: Unlocking the Secrets to Pancreatic Ductal Adenocarcinoma
Reported by Jessica Imperato, PhD. Candidate Molecular and Cellular Biology (MCB)
With millions of new cases diagnosed each year, cancer is the second-highest cause of death in the United States and is considered a major public health issue. While early detection is important, some types of cancers are much more difficult to identify, thereby lessening the effectiveness of conventional therapeutics. In particular, pancreatic cancer is problematic because of the organ’s position deep within the body and the delayed onset of symptoms. Consequently, pancreatic cancer is estimated to be the cause of approximately 7% of cancer-related deaths. A majority of pancreatic cancer cases are identified as pancreatic ductal adenocarcinoma. These tumors progress from lesions known as pancreatic intraepithelial neoplasias, or PanINs, which are formed after the transformation of pancreatic cells in a process known as acinar-to-ductal metaplasia (ADM). The formation of PanINs can be exacerbated by pancreatitis, as well as oncogenic mutations in the Kirsten rat sarcoma viral oncogene homolog ( KRAS). In addition, high expression of Kr üppel-like factor 5 (KLF5) has been shown in pancreatic tumors and appears to play a role in the survival of pancreatic cancer cells.
Here at Stony Brook University, Dr. Vincent Yang’s laboratory focuses on the role of KLF5 in the generation and progression of various gastrointestinal cancers and diseases. Recently, Dr. Ping He, an MCB graduate from the Yang lab published an elegant study in which he examined the role of KLF5 in PanIN formation and the progression of KRAS-induced pancreatic cancer. Upon inducing pancreas-specific oncogenic KRASin mice, Dr. He first observed KLF5 overexpression in the nuclei of cells obtained from PanIN lesions. This indicated that oncogenic KRAS may lead to elevated levels of KLF5, resulting in pancreatic lesions. KLF5-deficient KRAS mutant mice had fewer PanIN lesions after the induction of pancreatitis, further solidifying the necessity for KLF5 in the process of KRAS-mediated ADM and subsequent PanIN generation. Furthermore, Dr. He determined that in cases of oncogenic KRAS, downstream signaling pathways involved in cellular proliferation regulate and promote overexpression of Klf5. He also showed that the absence of Klf5resulted in the loss of activated STAT3, a transcription factor that has been associated with oncogenesis. Additionally, Dr. He demonstrated in vitro that the absence of KLF5 resulted in reductions in both cell proliferation and in the expression of pancreatic ductal markers in pancreatic cancer cell lines. These findings were reflected in mice implanted with tumors which were depleted of Klf5via shRNA. Mice whose tumors were depleted of Klf5experienced decreases in tumor size or complete tumor regression and had lower expression of pancreatic ductal markers within the tumors themselves ( Fig. 1).
Figure 1. Knockdown of Klf5 results in reduced size of pancreatic tumors.
Together, Dr. He’s findings showed that KLF5 plays an important role in cell proliferation and survival, the induction of ADM, and subsequent PanIN formation in the context of oncogenic KRAS .His findings will hopefully light the way for scientists and physicians to better understand the underpinnings of this devastating disease, leading the way to the development of improved therapeutics and clinical outcomes.
To read the full publication, please see:
He P, Yang JW, Yang VW, Bialkowska AB. Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice. Gastroenterology (2018). 154:1494–1508
Author Highlight: Dr. Ping He, PhD (MSTP)
One strand to rule them all: Zika virus manipulates host cells to infect neurons
Reported by: Arnav Choksi, Ph.D. candidate in Molecular & Cellular Biology (MCB)
First isolated from the Zika Forest of Uganda, Zika virus (ZIKV) has received much attention in the last decade due to its notorious and surprising spread over several continents. The virus’ ability to cross highly selective and secure biological barriers, such as the blood brain barrier (BBB) and the placenta, allows it to spread from an infected pregnant mother to her unborn child, causing microcephaly, where infants are born with small heads and under-developed brains. ZIKV infection can also cause Guillain-Barré syndrome in adults, resulting in progressive paralysis due to neuron damage. There is currently no vaccine or cure for ZIKV infection and not much is known regarding its mechanism of infection and pathogenesis.
Dr. Erich Mackow’s laboratory at Stony Brook University is interested in understanding how single-stranded RNA viruses alter cell signaling pathways to promote pathogenesis. Through her doctoral dissertation research, Ph.D. candidate Megan Mladinich has provided impressive insights into how Zika virus manipulates gene expression and signaling pathways in host cells. The ability of the virus to persist in human patients for up to 6 months in blood and other bodily fluids was a clear indication that the virus might be using certain cell types as reservoirs for persistence and subsequent infection of other cell types. Indeed, Megan found that ZIKV infects a subset of endothelial cells that form the blood brain barrier, called human brain microvascular endothelial cells (hBMECs). Interestingly, she found that exogenous IFN-α(Interferon-α; associated with antiviral activity), when added to hBMECs a few hours post infection with ZIKV, was unable to thwart infection, whereas prior addition of IFN-αsignificantly reduced the infection of hBMECs by ZIKV.This suggested that ZIKV infection alters hBMECs to resist IFN action. Moreover, while ZIKV infection is known to damage neurons and other cell types, hBMECs were not found to be damaged by ZIKV infection, suggesting that ZIKV infection followed a unique pathway in these cells. By analysing global transcription and cellular protein concentrations, Megan and her colleagues revealed that ZIKV-infected hBMECs significantly upregulate the expression of several factors that promote cell survival and prevent apoptosis. It was also found that following infection of hBMECs, ZIKV was released from the cells, potentially allowing ZIKV to infect neuronal compartments that are usually protected by hBMECs.
This clear and concise study offers a fundamental understanding of mechanisms that Zika virus might utilize to persist in specific cellular compartments. Moreover, it also sheds light on how the virus might manipulate host cells to establish and perpetuate infection in other cell types. Together, these data contribute to a pool of information that is necessary for the development of antiviral drugs to combat the spread of ZIKV infection.
To read the full publication, please see:
Author Highlight: Megan is an MCB Ph.D. candidate in the laboratory of Dr. Erich Mackow where she studies Zika virus persistence in endothelial cells that make up the blood brain barrier. Outside the lab, she enjoys indulging in long walks on the beach with her husband and dogs, eating snacks, hanging out with friends, and a good craft beer.
A needle in a haystack: The first identified degron sequence within an i-AAA protease substrate
Reported by: Julie Bettke, Ph.D. candidate in Molecular & Cellular Biology (MCB)
Mitochondria are multifunctional organelles that contribute essential cellular activities in eukaryotes, notably their role as the power generators of the cell. Maintaining the functional integrity of this organelle requires a dedicated network of protein quality control systems that regulate a proteome composed of ~1500 proteins distributed across two membranes (the inner and outer membranes) and two aqueous subcompartments (the intermembrane space and matrix). Alterations in the activities of these protein quality control systems compromise mitochondrial functions, and can result in the development of numerous neurodegenerative diseases. Therefore, it is critical to understand the processes that contribute to mitochondrial proteostasis.
Dr. Steven Glynn’s laboratory at Stony Brook University utilizes structural biology and biochemistry to deconvolute the mechanisms of ATP hydrolysis and proteolysis for mitochondrial AAA+ ( ATPases Associated with various cellular Activities) proteolytic machines. This family of proteases couples the energy of ATP hydrolysis to fuel the unfolding, translocation, and degradation of their substrates. Two such proteases, i-AAA and m-AAA, maintain mitochondrial protein quality control at the intermembrane space and matrix, respectively. To better understand how these proteases participate in mitochondrial protein quality control, the Glynn laboratory has engineered soluble active assemblies of both proteases to directly explore the process of substrate selection by these proteases. One mode of substrate recognition by AAA+ proteases is through sequence-specific elements within the substrate known as degrons. While several degron sequences have been identified for AAA+ protease family members, no such sequences have been identified for known substrates of the mitochondrial AAA+ proteases.
Dr. Anthony Rampello, a recent MCB graduate from the Glynn lab, identified the first degron recognized by the yeast mitochondrial i-AAA protease, Yme1p. He examined the degradation of two of its known substrates, Tim9 and Tim10, which function to deliver mitochondrial proteins to their proper subcompartments. He observed that Tim10 was degraded faster than Tim9, despite their high sequence and structural similarity. Mutational analysis revealed that a short phenylalanine-rich motif within the unstructured N-terminal region of Tim10 was necessary and sufficient to target the protein for degradation by Yme1p. Furthermore, Dr. Rampello found that other Tim proteins, including Tim12 and Tim13, contained similar motifs which marked them as targets for recognition and subsequent degradation by Yme1p. This study identified a degron within a native substrate of the yeast i-AAA protease and provides important insight into protease biochemistry.
To read the full publication, please see:
Rampello and Glynn (2017). Identification of a degradation signal sequence within substrates of the mitochondrial i-AAA protease
Author Highlight : Following his predoctoral work, Anthony joined the laboratory of Dr. Christian Schlieker in the Department of Molecular Biophysics and Biochemistry at Yale University. When not in the lab, Anthony enjoys reading, and dreams of visiting all the US National Parks.
Kamikaze specificity-enhancing factor for the AAA+ Lon protease
Reported by Padmina Shresth, PhD. Candidate Molecular & Cellular Biology (MCB)
AAA+ proteases are ATP-fueled molecular machines that work like a wood chipper to get rid of unwanted proteins. Conserved from bacteria to humans, Lon protease regulates protein homeostasis in mitochondria, and altered Lon activity has been implicated in several diseases such as CODAS syndrome and cancer. Hence, understanding Lon biology is important in order to design therapeutics. Here at Stony Brook University, Dr. Wali Karzai’s lab is exploring mechanisms of Lon regulation in regard to directed proteolysis. MCB student Neha Puri, a former member of the Karzai lab, discovered a novel mechanism by which heat shock protein Q (HspQ) regulates the selective degradation activity of Lon in the pathogenic bacterium Yersinia pestis. Interestingly, Neha discovered that HspQ is the founding member of a novel class of protease adaptors in that it functions both as a substrate and a specificity-enhancing factor for Lon.
Lon is known to target folded, functional and native proteins to regulate cellular processes, such as cell cycle arrest under stressful conditions. In a recent publication, Neha reveals that HspQ enhances the ATP hydrolysis rate of Lon and its proteolytic activity for certain protein substrates. However, unlike other specificity-enhancing factors, HspQ is degraded in the process. Elegant biochemical assays classified HspQ as an allosteric activator of Lon. Neha constructed several mutants of HspQ to show that its C terminus is necessary and sufficient for Lon to recognize it as a substrate, but a second site of interaction in its native core is also required for allosteric activation of Lon.
Maintaining a balance of Lon expression and activity is important for protein homeostasis. Hence, knowledge about the mechanism of substrate selection and degradation by Lon is helpful for designing drugs to target specific Lon activities that cause diseases, without affecting its critical regulatory function.
To read the full publication, please see:
Puri, N & Karzai, W. (2017). HspQ functions as a unique specificity-enhancing factor for the AAA+ Lon Protease. Molecular Cell 66:672-683.
Author Highlights: Neha Puri
Neha graduated from the MCB program from the laboratory of Dr. Wali Karzai in December 2016. Her thesis research discovered and characterized a novel accessory protein for the bacterial Lon protease. After completing her Ph.D., she joined the laboratory of Dr. James Berger at the Johns Hopkins School of Medicine to learn biophysical and structural biology tools to answer questions pertaining to complex biological machineries involved in E. coli DNA replication initiation. When she is not in the lab, she enjoys hiking, gardening, optimizing shortcuts to really long recipes, and reading fiction.
One Big Problem: A New Function for Dystrophin the Largest Human Gene
Reported by Megan Mladinich, PhD. Candidate Molecular & Cellular Biology (MCB)
Duchenne muscular dystrophy (DMD), a fatal muscle disease caused by mutations in the dystrophin gene, currently affects 300,000 boys worldwide. DMD patients experience extensive muscle degeneration and weakness. Additionally, large subsets of DMD patients also have neurodevelopmental deficits including smaller brain volume and cognitive impairments. Dystrophin is a component of protein complexes that connect the cellular cytoskeleton and extracellular matrix to foster plasma membrane integrity and cellular signaling. Dystrophin complexes, although best characterized in muscle, have also been detected in the brain and loss of dystrophin has been shown to delay myelination of the brain. Myelination, the process of coating neurons with a myelin sheath, is critical for the central nervous system (CNS) to transmit signals at maximum speed and efficiency. Oligodendroglia are the supporting cells responsible for myelination of neuronal cells and proper maturation of oligodendrocytes is crucial for normal brain function. The Colognato Laboratory here at Stony Brook investigates the influence of niche extracellular matrix proteins on developing oligodendroglia and neurodevelopment. Specifically, the Colognato Lab is interested in advancing knowledge of the role of dystrophin and associated complexes on oligodendrocytes, the myelinating cells of the CNS.
In a recent publication, MCB alum Dr. Azeez Aranmolate and colleagues characterized the oligodendrocyte dystrophin complex for the first time. Using both mRNA expression and immunohistochemistry, Azeez showed that dystrophin and associated protein complexes were expressed in developing oligodendrocytes. Analysis revealed that oligodendrocytes expressed three dystrophin isoforms, Dp427, Dp140 and Dp71 (Figure 1) and that dystrophin isoform expression and cellular localization was developmentally regulated. Dp427 and Dp140 were the predominant isoforms in immature oligodendrocytes, while Dp71 was prominent in mature myelin-competent oligodendrocytes. Furthermore, mdxmice, which model DMD due to a lack of Dp427, displayed a developmental myelination delay in the cerebral cortex likely due to an oligodendrocyte requirement for dystrophin. Finally, primary oligodendrocyte cultures were found to require dystrophin for timely and appropriate maturation, and loss of dystrophin led to a significant delay in myelin protein production.
Previously, the cellular basis for neurodevelopmental disorders in DMD patients was poorly understood. Together, the results obtained by Dr. Aranmolate and colleagues indicate that improper oligodendrocyte maturation and myelination has the potential to contribute to the CNS developmental delays seen in DMD patients. These findings provide new insights into potential cellular contributors and molecular pathways involved in DMD brain dysfunction. Future studies will be needed to better understand dystrophin’s role in these and other cellular processes in pursuit of pharmacological strategies to enhance myelination and myelin repair in a damaged CNS.
To read the full publication, please see:
- Aranmolate et. al., Myelination is Delayed During Postnatal Brain Development in the MDX Mouse Model of Duchenne Muscular Dystrophy
Author Highlights: Azeez Aranmolate
In addition to his passion for science, Azeez also enjoys beaches, riding motorcycles, fitness, trying new foods and traveling with friends. Azeez is currently a Postdoctoral Scholar in the Department of Microbiology, Immunology & Molecular Genetics at University of California, Los Angeles (UCLA). Thus, when he’s not busy beaching it up or doing motorcycle runs up and down the Pacific Coast Highway, he can be found studying mechanisms of infection between Trichomonas vaginalis (Tv) and human host cells in-vitro and in-vivo. Lastly, Azeez was recently awarded a NIH-IRACDA (Institutional Research and Academic Career Development Award) by the UCLA's Postdocs Longitudinal Investment in Faculty Training (UPLIFT) Program. Accordingly, starting in fall of 2018, as a NIH-IRACDA Postdoctoral Fellow, Azeez will dedicate most of his time to biomedical research, but additionally receive advanced pedagogical training at UCLA and a teaching practicum in college level instruction at California State University, Los Angeles (CSULA).
Something Fishy is Going on in the Martin Lab: FGF and canonical Wnt signaling cooperate to induce paraxial mesoderm from tailbud neuromesodermal progenitors through regulation of a two-step epithelial to mesenchymal transition
Reported by Anthony Rampello, Ph.D. candidate in Molecular and Cellular Biology (MCB)
Developmental biology details how the temporal coordination of signaling pathways that dictates the various processes required for the growth of a single cell into complex, multicellular organism. Interestingly, the molecular pathways that regulate embryonic development are the same pathways that are associated with human diseases. Thus, deciphering the mechanisms controlling embryonic development provides us with a better understanding of not only developmental defects, but also pathogenesis of other diseases, such as cancer. Current efforts in the laboratory of Dr. Benjamin Martin take a molecular approach at describing the genetic networks regulating development using zebrafish embryos as model systems. Zebrafish embryos develop ex utero and their optical transparency permits live imaging. A particular interest of the Martin lab involves studying the molecular mechanisms that regulate the induction and patterning of mesoderm, a germ layer, which gives rise to skeletal muscle tissues and vasculature. In a recent publication, graduate student Hana Goto and colleagues describe the role of one such mesoderm-promoting signaling pathway, fibroblast growth factor (FGF) signaling pathway. They report that, throughout development, the role of FGF changes from inducing mesodermal fate commitment during gastrulation (earlier developmental stage) to regulating the cell movement during post-gastrula stage (later developmental stage). Precisely, FGF is essential for epithelial to mesenchymal transition (EMT), a process implicated in not only embryonic development, but also in cancer metastasis and wound healing.
Figure 1: Model for multistep mesodermal EMT.
(i) Wnt signaling initiates EMT in brachyury/ sox2 expressing NMPs; (ii) cells become mobile and begin expressing vimr1 and vimr2; and (iii) FGF-mediated termination of EMT via tbx16 and msgn1 expression resulting in the formation of paraxial mesoderm.
During post gastrula stages, the posterior body tissues develops from a small group of bipotential progenitor cells known as neuromesodermal progenitors (NMPs). NMPs give rise to both spinal cord and mesoderm. Although FGF is known to be critical for mesoderm development from NMPs, the mechanism of how FGF promotes posterior mesoderm was unknown. Through the use of inducible transgenic embryos and high-resolution imaging, Hana and colleagues found that during post gastrula stages, FGF promotes the completion of EMT to establish mesoderm. Their studies identified two vimentin-related proteins (Vim1 and Vim2) as EMT-specific biomarkers that allowed for an unprecedented visualization of cell migration during mesodermal EMT. Strikingly, Hana and others determined that FGF signaling indirectly represses two progenitor genes that mark NMPs ( sox2 and brachyury) through the activation of maturation genes, tbx16 and msgn1. This signaling pathway results in not only EMT termination but also maturation of progenitors into mesoderm. Based on these findings, Hana and colleagues propose a new model outlining the multistep mesodermal EMT (Fig. 1).
Previously, visualization of EMT was limited to in vitro and ex vivo cell culture studies. The findings presented by Hana and her colleagues show that NMPs could be utilized as an in vivo model to study EMT. It is interesting to think what additional factors are involved in these developmental processes, as well as their potential as therapeutic targets in human disease. For instance, brachyury has been found to be associated with aggressive breast cancer and hepatocellular carcinoma. In addition, Hana and colleagues hope that by studying the regulatory network of NMPs, their research will be useful in developing stem cell based therapies for spinal cord and muscular-skeletal defects.
To read the full publication, please see:
Goto, H., et al., FGF and canonical Wnt signaling cooperate to induce paraxial mesoderm from tailbud neuromesodermal progenitors through regulation of a two-step epithelial to mesenchymal transition. Development, 2017. 144(8): p. 1412-1424.
Hana is a fifth year Ph.D. student in the laboratory of Dr. Benjamin Martin. When she's not in the lab, Hana enjoys traveling, running, and anything food related (cooking, baking, eating out, going to farmer's market).
Suppressor of TCR signaling (Sts-1, and -2) knockout mice offer insight on host-virus
Reported by Nadia Jaber, Ph.D. candidate in Molecular and Cellular Biology (MCB)
Emerging interests in host-virus response among Stony Brook investigators Dr. Laurie Krug and Dr. Nicholas Carpino has brought about a successful scientific collaboration. Dr. Carpino studies the role of Suppressor of TCR signaling -1 and -2 (Sts-1 and -2) and discovered that genetic deletion of this suppressor leads to hyper-activation of T-cells. Dr. Krug’s lab is interested in gammaherpesvirus, which establishes life-long infections and can lead to serious malignancies. Gammaherpesviruses establish latency in host cells for many years before reactivating and manifesting an infection. T-cells have a wellappreciated role in controlling gammaherpesvirus infections during both the acute and latent phases of infection.
Graduate student Brandon Cieniewicz characterized the Sts-1/Sts-2 double knockout (dKO) mice infected with murine gammaherpesvirus 68, which has pathological similarities to human disease. Brandon found that T-cells from Sts dKO mice increased interferon-γ production (Figure 1), and degranulation in response to infected cells. Brandon hypothesized that the enhanced activity of T-cells in Sts dKO mice may increase clearance of the gammaherpesvirus infection. In addition, the dKO Tcells may require less stimulation for activation and may be better equipped to recognize latent infections. Contrary to his hypothesis, Brandon found no effect of Sts dKO on acute viral replication, establishment of latency, or reactivation from latency in vivo. He observed increased levels of total CD8 + T-cells in Sts dKO, but not of the virus-specific pool.
Brandon and colleagues concluded that deletion of Sts-1 and -2 does not confer a greater ability to clear gammaherpesvirus infections. Although the Sts molecules are implicated for their role in T-cell receptor signaling, it is plausible that they play a similar role in other signaling pathways, and that its deletion leads to effects that offset the hyper-activity of T-cells. The investigators believe a CD4 + or CD8 + T-cell-specific deletion may offer more insight. Still, the investigators believe the hyper-responsive Sts dKO T-cells could be harnessed for therapies involving adoptive transfer, such as cancer immunotherapy. In addition, Brandon thinks the Sts dKO system could be used to study other infectious diseases. Another collaboration with the Konopka lab shows promising results for the clearance of yeast infections.
To read the full publication, please see
Cieniewicz B, Carpino N, Krug LT (2014) Enhanced Response of T-Cells from Murine Gammaherpesvirus 68-Infected Mice Lacking the Suppressor of T-Cell Receptor Signaling Molecules Sts-1 and Sts-2 . PLoS ONE 9(2): e90196.
Brandon is a sixth year Ph.D. student in the laboratory of Dr. Laurie Krug. When he’s not in the lab, Brandon spends his time brewing beer for the annual Oktoberfest party, trying new vegetarian recipes and hiking in the woods.
A novel role for cytosolic Phospholipase Cβ in reversal of siRNA hydrolysis by C3PO
Reported by: Yang Liu, Ph.D. Candidate Molecular and Cellular Biology (MCB)
Phospholipase Cβ (PLCβ) 1-4 is a family of Gα downstream signaling enzymes that elicit increased levels of intracellular calcium and mediate PKC activation. Although the majority of the PLCβ resides on the plasma membrane where it interacts with Gαq , the function of the cytosolic pool of PLCβ is elusive. Recent work in the laboratory of Dr. Suzanne Scarlata showed that cytosolic PLCβ strongly binds to the endonuclease translinassociated factor X (TRAX) and reverses siRNA mediated down regulation by interfering with the function of the RNA silencing promoter C3PO. In a recent publication in the Journal of Biological Chemistry, graduate student Shriya Sahu and her colleague Dr. Finly Philip, characterized how the association between PLCβ and C3PO affects the hydrolysis of certain siRNAs.
While PLCβ reverses silencing by siRNAs against GAPDH but not Hsp90, the explanation for such a phenomenon is unclear. To rule out the possibility that it is merely due to differences in the compartmentalization of the cytosolic PLCβ pool and TRAX population, Sahu visualized fluorescently labeled proteins in the cytosol using FRET, and identified the consistent association of PLCβ with TRAXcontaining C3PO complexes. By analyzing the aggregation states of the purified translin, TRAX and C3PO, the authors showed that C3PO is octameric in solution. Further study of the association of PLCβ with C3PO complex using FRET, suggested a model in which a single molecule of PLCβ binds to C3PO on an external site on the TRAX subunits without affecting the assembly of C3PO complexes (Figure 1).
To study how PLCβ affects C3PO’s function, they measured the displacement of GAPDH and Hsp90 siRNAs labeled with a fluorescence probe from C3PO and C3PO-PLCβ with the addition of increasing amounts of ssDNA. Interestingly, they found that siRNA (Hsp90) binds to C3PO complexes more strongly than siRNA (GAPDH), even though PLCβ slightly weakened the binding of the both siRNA species. Sahu and coworkers proposed that the differences in the binding reflect differences in nucleotide structure. Fluorescence quenching studies supported this idea by suggesting a less stable structure of Hsp90 siRNA. However similar protection was seen for both siRNAs on binding to C3PO, and similar decreases in protection were seen in the presence of PLCβ. These results suggest that the effect of PLCβ on C3PO function may lie in the nature of the microRNAs. If this is the case, then differences in the effect of PLCβ on C3PO-mediated siRNA degradation could be due to the differences in siRNA’s vulnerability to hydrolysis. When they measured the hydrolysis rates of siRNAs by C3PO, they consistently observed that GAPDH siRNAs was hydrolyzed faster than Hsp90 siRNAs and PLCβ binding only reduced the rate of hydrolysis of siRNA (GAPDH) but not siRNA (Hsp90). Therefore, the reason why PLCβ reverses degradation by siRNA(GAPDH) but not the Hsp90 could be because of intrinsic differences in hydrolysis by C3PO.
This work supports the existence of a novel function of cytosolic PLCβ in RNA interference and gene regulation in addition to its traditional role in generating calcium response by G-protein signaling. Moreover, the molecular mechanism underlying the function of PLCβ in reversal of siRNA mediated gene silencing adds another interesting dimension to the way gene expression might be controlled.
To read the full article, please see:
Sahu, S, et al. (2014). Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ. Journal of Biological Chemistry 289(8): 5134-5144.
Shriya is a PhD candidate in the MCB program and works in the laboratory of Dr. Suzanne Scarlata at Stony Brook University. Her thesis research involved characterizing the interaction of PLCβ with C3PO and uncovering the molecular mechanism of C3PO nuclease activity. Aside from scientific research, Shriya’s interests include traveling to new places, hiking, dancing and cooking.
Lysosomal protease inhibitor helps tumor cells deal with stress
Reported by: Jennifer L. DeLeon, Ph.D. Candidate Molecular and Cellular Biology (MCB)
Squamous cell carcinoma antigen, or SCCA, was identified as a serum biomarker in several cancer types including cervical, lung, head and neck, and liver. However, the mechanism by which upregulation of SCCA contributes to tumorigenesis has been poorly understood. Recent work in the laboratory of Dr. Wei-Xing Zong showed that SCCA overexpression is associated with poorly differentiated and aggressive cancers of the breast, colon and pancreas. In normal tissue, SCCA functions as a protease inhibitor to block cytosolic leakage of lysosomal proteases and promote cell survival in the event of lysosomal damage. MCB alum Dr. Namratha Sheshadri and colleagues uncovered a tumorpromoting role of SCCA, which was recently published in Cancer Research. Dr. Sheshadri discovered that the upregulation of SCCA promotes the unfolded protein response and activation of pro-inflammatory signaling through the cytokine IL-6 in cell lines and a mouse model of mammary tumorigenesis.
|Figure 1: SCCA promotes EMT|
To study the tumor promoting function of SCCA, Sheshadri et al. overexpressed SCCA in the “normal” non-neoplastic human breast epithelial cell line MCF10A. Upon SCCA overexpression, they observed phenotypic changes resembling epithelial to mesenchymal transition (EMT); a change of cultured cells from a cobblestone-like monolayer to a spindle morphology (Figure 1). Likewise, transcription analysis showed an increase in EMT promoting markers like ZEB1 and vimentin, and the loss of epithelial markers like E-cadherin. SCCA overexpression leads to increased migration, anchorage independent colony formation, and growth factor autonomous cell proliferation and survival.
To elucidate the molecular mechanism for the cellular EMT like phenotype, Sheshadri screened several EMTpromoting growth factors and identified a significant increase in IL-6 transcription and secretion in SCCA expressing cells. Interestingly, pharmacological inhibition or genetic knockdown of IL-6 abrogated several of the transformation phenotypes induced by SCCA. IL-6 has been well appreciated as a potent cytokine for cell signaling, inflammation, and tumorigenesis, however, the question still remained, how does SCCA promotes IL-6 expression?
In a previous study, SCCA overexpression was shown to block lysosomal and proteasomal protein turnover, thus disrupting normal protein homeostasis. Sheshadri hypothesized that SCCA may potentiate the build-up of misfolded and ubiquitinated proteins and trigger the cellular stress response termed the unfolded-protein response (UPR). Indeed, SCCA transformed cells have increased levels of several UPR markers and display a chronic but nonlethal endoplasmic reticulum stress response. By genetic knockdown of these ER stress receptors, Sheshadri demonstrated a decrease in IL-6 transcription. Thus, the impaired protein degradation caused by elevated SCCA expression may lead to UPR, which has been implicated in pro-inflammatory response in numerous human diseases including cancer.
This work establishes an important example where disruption of protein homeostasis can contribute to EMT and tumorigenesis via the unfolded protein response. Importantly, it also identifies the Achilles heel for tumors that exhibit high expression of SCCA such that they are sensitized to ER stress-inducing pharmacological agents; and thus may be an advantageous therapeutic target in human cancers.
To read the full article please see:
Sheshadri, N., et al. SCCA1/SERPINB3 Promotes Oncogenesis and Epithelial-Mesenchymal Transition via the Unfolded Protein Response . Cancer Research. 2014 Sep 11.
Dr. Namratha Sheshadri, Ph.D.
Namratha graduated from Stony Brook University with a Ph.D. in Molecular and Cell biology from the laboratory of Dr. Wei-Xing Zong in August 2014. Her thesis research uncovered the cellular mechanism of lysosomal protease inhibitor SCCA to initiate breast cancer tumorigenesis. Namratha recently obtained a post-doctoral position at the National Cancer Institute (NCI). Aside from scientific research, Namratha’s interests include bird watching, gardening, hiking and traveling.