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Student News and Views

August 2017

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.

Author Highlights: Hana Goto
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).

February 2015

Suppressor of TCR signaling (Sts-1, and -2) knockout mice offer insight on host-virus response
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.

Cieniewicz Figure 1Graduate 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.

Brandon CieniewiczTo 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.

Author Highlight: Brandon Cieniewicz
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.



November 2014

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.

Sahu Figure 1While 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.

Shriya SahuTo 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.

Author Highlight: Shriya Sahu
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.


October 2014

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
  Figure 1

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.

Dr. Namratha SheshadriTo 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.

Author Highlight: 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.



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