Our Research Projects

 

GTP metabolism in melanoma cell invasion 

Guanylate Metabolism Enzymes
Recently, we have reported a fundamental connection between RHO-GTPase activity and GTP metabolism enzymes (GMEs) (Wawrzyniak et al. Cell Reports 2013). In particular, we demonstrated that in melanoma cells restoration of normal melanocyte levels of GMPR (see Fig) or partial inhibition of IMPDH led to a moderate ~25% depletion of intracellular GTP pools and a dramatic drop in the amounts of GTP-bound i.e. active RAC1. As a result, melanoma cell invasion was significantly downregulated. Restoration of GTP levels reverted these effects. Additionally, GMPR overexpression or inhibition of GMPS, a functional antagonist of GMPR, suppressed melanoma cell tumorigenicity (Bianchi-Smiraglia et al. Cell Death and Differentiation 2015; Oncogene 2017).


Historically, changes in GTP levels were not considered as a regulatory step in activation of RAC1 (or other G-proteins) in live cells. This is because average GTP concentration in the cell measured by HPLC (~500µM)  is much higher than the GTP dissociation constant of RAC1, which even in presence of GEF, is ~20µMGTP. However, HPLC cannot account for free vs bound GTP or for sub-cellular variations in GTP levels. Up until now no methods existed to detect such variations. We recently reported genetically encoded intracellular sensors of free GTP (Bianchi-Smiraglia et al. Nature Methods, 2017). These sensors for the first time made possible visualization of GTP changes in live cells and identified regions with low (~30µM) and high local GTP concentration. Currently, we are studying mechanisms underlying the role of guanylate metabolism in melanoma invasion and metastasis and characterizing anti-guanylate therapy as novel strategy for treating melanoma.

MP1 16um Spatiotemporal variation

Exploring lineage-specific transcriptional regulators of tumor progression

carcinoma vs melanoma

Identification of lineage-specific transcription factors regulating tumor progression is important for understanding vulnerabilities of a specific cancer type. Of a particular interest are transcription factors oppositely regulating transformed phenotypes in different tumor lineages. 

In epithelial cells, E/N cadherin switch represents a hallmark of epithelial-mesenchymal transition (EMT), a process by which static cells acquire a mesenchymal-like phenotype, including migratory and invasive capabilities. In search for transcription factors regulating the EMT-like transition in melanoma cells, we evaluated several bona fide regulators of EMT. One of them, FOXQ1, a member of FOX family of transcription factors, is overexpressed at advanced stages in several human carcinomas where it promotes E/N-cadherin switch ultimately leading to increased invasion. 
Unexpectedly, we found that FOXQ1 levels are significantly lower in metastatic melanoma specimens compared to primary melanomas. Accordingly, we identified that in melanoma cells, FOXQ1 suppresses the same processes it activates in carcinoma cells: expression of N-cadherin gene (CDH2), EMT, invasion, and metastasis.  Mechanistically, we demonstrated that like LEF1/TCF4, FOXQ1 interacts with nuclear -catenin and TLE (groucho) proteins and that the ß-catenin/TLE ratio, which is higher in carcinoma than melanoma cells, determines the effect of FOXQ1 on N-cadherin gene transcription.  Accordingly, other FOXQ1-dependent phenotypes can be manipulated by altering nuclear ß-catenin or TLE proteins levels (Bagati et al. Cell Reports 2017) (see Fig). Intriguingly, FOXQ1 is also involved in promotion of differentiation in both normal epithelial and melanocytic cells (Bagati et al. Cell Death and Differentiation 2018) via unknown mechanisms. Currently, we are establishing such mechanisms and in parallel pursuing the role of Foxq1 deficiency in promotion of melanomagenesis in mouse models of melanoma.

Novel metabolic targets in multiple myeloma 

no cell death vs cell death
A
. It is well known that high ROS levels increase nuclear amounts of NRF2, a universal transcriptional activator of anti-oxidant genes. Recently, we have for the first time reported that under certain conditions, NRF2 can paradoxically amplify ROS (Zucker et al. Molecular Cell 2014). In particular, we demonstrated that when ROS levels exceed a certain threshold, NRF2 induces expression of the transcription factor KLF9 (Kruppel-like factor 9). KLF9 represses several anti-oxidant genes, which are not NRF2 targets, including mitochondrial thioredoxin reductase (TXNRD2), thus resulting in ROS amplification (see Fig). TXNRD2 protein is critical for maintenance of intracellular red-ox status and ROS detoxification. 

B. Intriguingly, we have also identified for the first time that KLF9 plays an important role in multiple myeloma (MM) drug resistance (Mannava et al. Blood 2012). Multiple myeloma is a plasma cell disorder that accounts for approximately 10% of all hematologic malignancies. Although the introduction of novel agents in the past decade has increased the median overall survival of myeloma patients from 30 months to 45-72 months, the disease remains incurable. One such agent, proteasome inhibitor bortezomib (Velcade®, PS-341), significantly increased overall survival in patients with relapsed or refractory multiple myeloma. Induction of endoplasmic reticulum (ER) stress and subsequently, ER stress response constitutes one of the major pathways of bortezomib cytotoxicity. Although low levels of ER stress are aimed at elimination of unfolded proteins, unresolved or high-level ER stress triggers pro-death pathways. 

It has been generally accepted that intracellular oxidative stress induced by proteasome inhibitors is a byproduct of endoplasmic reticulum (ER) stress. We demonstrated a mechanism underlying the ability of proteasome inhibitor bortezomib or its next generation analog carfilzomib to directly induce oxidative and ER stresses in multiple MM cells via transcriptional repression of TXNRD2 gene (Fink et al. Leukemia 2016). These finding identify novel mechanism of regulation of oxidative stress and identify a novel target for MM therapy.

C. Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of xenobiotic response. Our study revealed that AHR also positively regulated intracellular polyamine production via direct transcriptional activation of two genes (ODC1 and AZIN1) involved in polyamine biosynthesis and control, respectively. In multiple myeloma patients, AHR levels inversely correlated with survival, suggesting that AHR inhibition may be beneficial for treatment of this disease We identified clofazimine, an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of multiple myeloma (Vk*Myc mice) and in SCID mice bearing multiple myeloma cell xenografts, revealed high efficacy of clofazimine comparable to that of bortezomib, a first-in-class proteasome inhibitor used for treatment of multiple myeloma (Anna Bianchi-Smiraglia et al. Journal of Clinical Investigations 2018).  Currently, we evaluate the role of TXNRD2, AHR and KLF9 in MM resistance to chemotherapeutic agents and pursue mechanisms of activation of KLF9 by proteasome inhibitors.