postheadericon Researh

Cell commitments to grow in size, differentiate, proliferate, transform or initiate cell cycle arrest, DNA repair, and then survive or undergo apoptosis if the damage is beyond repair, are guided by the unique assortment of external cues found in local microenvironment that activate complex receptor-mediated intracellular signaling networks. Despite a rapid growth of our knowledge of protein and lipid components involved in these multilayered cell signaling networks, an integrative, quantitative picture of their dynamic behavior and inter-pathway cross-talk remains elusive.

As experimental cell biology researcher, I belong to the Systems Biology of Cell Signaling Networks group, which combines computational methods and experimental data to understand how cells operate as integrated molecular systems and interpret the signals in a context-dependent manner, which is important for development of strategies to effectively target tumor cells with novel molecular therapeutics. To achieve this long-term goal, my research has been primarily focused on the dynamics of epidermal growth factor (EGF) receptor signaling and its interaction with hormone/cytokine signaling networks in normal and neoplastic cells.

Abnormal expression and activity of growth factor, hormone and cytokine receptors as well as various molecular components of oncogenic signaling pathways is common in different solid tumors that display distinct drug sensitivity profiles. Depending on cellular context, these pathways can either stay individual and signal in parallel or may interact (cross-talk) with each other in a positive and/or negative manner at different stages of signal propagation that may readily contribute to intrinsic and/or acquired chemoresistance, which, in turn, is a major cause of treatment failure as well as poor prognosis in various human malignancies.

My postdoctoral research

Receptor tyrosine kinase signaling

My graduate work, which has been performed in collaboration with clinical researchers to determine the genetic polymorphism of human apoplipoprotein E (apoE) among cardiovascular patients in Kaunas city (Lithuania) by using CAPS analysis, evoked my genuine interest in ApoER2 receptor signaling and, in general, in Signal Transduction.

In order to gain more knowledge in molecular signaling mechanisms underlying important human disorders, I joined the Systems Biology of Cell Signaling Networks Laboratory at Thomas Jefferson University (Philadelphia, PA), conducting research on Receptor Tyrosine Kinase (RTK) signaling networks.

Since then, I obtained and analyzed quantitative information on expression and spatio-temporal activation patterns of kinases, phosphatases and adaptor proteins that belong to cell survival, motility, growth and differentiation signaling pathways, placing a special emphasis on their hierarchy in the network, feed-forward and feedback regulations, cross-talk and its role in cancer cell proliferation, viability as well as resistance to anti-cancer agents. Small molecule inhibitors and RNA interference-based systematic perturbations allowed me to delineate the nature and properties of interactions between the distinct signaling cascades downstream of activated EGF, prolactin and insulin receptors, as well as to investigate the roles of Insulin-related substrate (IRS) family adaptor proteins in fine-tuning of the amplitude and duration of the cytoplasmic signaling responses, which are subsequently channeled into dynamic nuclear response patterns of immediate-early genes that shape cell-fate decisions.

Improved data acquisition for analysis of network architecture

Extensive qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their roles in diverse cellular processes. Conventional immunoblotting procedure, through sensitive, is prone to produce substantial errors and is not readily adapted to rapid acquisition of large amount of data. Therefore, first of all, I developed higher-throughput Multi-strip Western Blotting (MSWB) method, which allows monitoring of up to nine different proteins and results in up to ten-fold increase of data points generated per single blotting cycle. In addition, MSWB substantially improves data accuracy and allows concomitant comparison of signals derived from multiple blots.

Context-dependent cross-talk between survival and mitogenic pathways
IRS family adaptor proteins as therapeutic targets

My experimental results helped to demonstrate the bidirectional reciprocal cross-talk between the cell survival (PI3-kinase/Akt) and mitogenic (Ras/Raf/MEK/ERK) signaling pathways and revealed a critical role of Grb2-associated binder 1 (GAB1) as an integrating node of PI3-kinase/Akt and Ras/ERK signaling upon EGF stimulation, which confirmed the predictions of a mathematical model.

Further studies were focused on involvement of adaptor proteins in numerous interactions that propagate signaling by EGF and insulin, showing that insulin-induced amplification of mitogenic Ras/MAPK signaling is abolished by disrupting the PI3-kinase product's phosphatidylinositol (3,4,5)-trisphosphate PIP3-mediated positive feedback via GAB1 and IRS1. I also showed, that siRNA-mediated suppression of GAB1 protein levels markedly increased the cytotoxic effects of 5-fluorouracil in K-Ras mutant pancreatic cancer cells, suggesting that GAB1, and perhaps other IRS family members, has a potential value as therapeutic targets for this incurable type of cancer.

I assumed that dynamic PI3-kinase-ERK interactions also persist in tumors, making the activation patterns robust to single versus combinatorial treatments, which can be linked to different drug sensitivity profiles. In a proof of principle experiments I demonstrated that the cells without or weak ERK1/2-mediated negative feedback towards PI3-kinase (e.g. MCF-7 breast cancer cells) are more sensitive to growth inhibition by MEK inhibitor U0126 compared with those, that provide a strong feedback upon EGF stimulus (e.g. A431 epidermoid carcinoma). In contrast, the combined targeting of PI3-kinase and MEK/ERK signals synergistically decreased cell viability and chemosensitized cancer cells to growth-inhibitory treatments, underlining a novel paradigm for breast cancer therapy.

Our work with different types of cancer cells has provided much evidence that the cross-talk between two different signaling modules and individual protein contribution is not at stiff or stable state, but dynamically changes, depending on the ligand type, its dosage (signal strength) and stimulation time. These observations were summarized in a recent review. I strongly believe that quantitative understanding of signaling mechanisms does not favor restrictions to one cell type, and suggest that detailed protein activation/deactivation and/or expression patterns should be assessed in cells from diverse tissues upon various stimulus strenght and duration. Only then we would be able to make more consistent conclusions about signaling specificities and find out whether the molecular signatures indeed differ tremendously across malignancies.

Quantitative characterization of prolactin receptor signaling network

The pituitary lactogenic hormone and pro-inflamatory cytokine prolactin (PRL) participates in an autocrine/paracrine stimulatory loop within breast tissues and is required for the terminal differentiation of normal human breast. Compared to normal tissue, mammary tumors express higher amounts of PRL Receptor, which promotes proliferation, survival and migration of cancer cells, contributes to angiogenesis, and thus plays an essential role in pathogenesis of breast cancer. PRL-mediated activation of JAK/STAT and MAPK pathways has been reported to antagonize cytotoxicity of anti-cancer drugs taxol, doxorubicin and cisplatin.

However, the signaling via PRL receptor has not been examined extensively in a quantitative manner. Therefore, I applied systematic and controlled perturbation experimental analysis to define the flow of signaling information through the JAK/STAT, Src/FAK, PI3-kinase/Akt, Rac/PAK and Ras/MAPK signaling branches and assessed the contribution of PI3-kinase/Akt towards MAPK (Raf/MEK/ERK) pathway activation in T47D and MCF7 breast cancer cells. The results revealed the uncommon predominance of PI3-kinase/Akt-dependent Rac/PAK signaling over the canonical Shc-Grb2-SOS-Ras route in activation process of ERK1/2 kinases, which may have an impact when choosing and implementing suitable treatment strategies.

MEK-independent ERK1/2 activation

I was co-leading a project, aimed to describe an activated ERK1/2 resistance to MEK inhibition/suppression in PI3-kinase-mutant estrogen receptor (ER)-positive T47D (and to a lesser extent in BT-474) breast cancer cells in the ErbB family, but not other growth factor, signaling environment, which, nevertheless was sensitive to PI3-kinase/Akt functioning, and may underlie higher survival capability and anti-estrogen therapy resistance. We discussed the candidate proteins that could be involved in this activation circuitry, performed RNAi screening and provided first evidence that PBK/TOPK serine/threonine kinase may play a role in mediating MEK-independent ERK activation

Bimodal Cell Population Responses Can Emerge from the Interplay Between Analog Single-Cell Signaling and Protein Expression Noise

We applied biochemical measurements and deterministic and probabilistic models to study how network topologies and cell-to-cell protein abundance variations shape signaling outcomes in the context of EGF-induced ERK1/2 activation in HEK293 cells. We observed bimodal ERK activation response distributions in cell populations, which are thought to imply bistable or ultrasensitive signaling behavior in single cells. Surprisingly, however, we found that an analog RasGTP input to a simple MAPK cascade model with a negative feedback, which generates smooth single-cell dose responses, can explain the bimodality in our data. Model analysis suggests that such a conversion of smooth input-output responses in single cells to digital responses at the population level can be caused by protein abundance variability, which gives rise to a wide distribution of ERK pathway activation thresholds. Thus, cells can keep the well-known robustness benefits offered by a negative feedback system, while simultaneously generating population-level on/off responses that are thought to be critical for cell fate decisions.

Adiponectin fine-tuning of liver regeneration dynamics revealed through cellular network modeling

Following partial hepatectomy, the liver initiates a regenerative program involving hepatocyte priming and replication driven by coordinated cytokine and growth factor actions. We investigated the mechanisms underlying adiponectin's (Adn) regulation of liver regeneration through modulation of these mediators. We developed a computational model describing the molecular and physiological kinetics of liver regeneration in Adn-/- (knockout) mice.

Pro-apoptotic, cytostatic and chemosensitizing effects of PI3-kinase-targeting bioflavonoids in breast and pancreatic cancer cells

I was engaged in the studies that extend our knowledge about the short-, medium- and long-term signaling responses of cancer cells exposed to acute and chronic treatment with natural dietary bioactive compounds with anti-cancer potential. Specifically, I explore the effects and mechanisms of action of bioflavonoid luteolin (natural inhibitor of PI3-kinase) on breast and pancreatic cancer cell growth, viability and sensitivity to established chemotherapy drugs 5-fluorouracil (Adrucil) and Bcr-Abl inhibitor nilotinib (Tasigna) in the presence or absence of EGF and IGF-1, two potent mitogens that drive breast and pancreatic tumor progression. This project is important in searching for alternative synergizing therapeutic approaches to overcome intrinsic and acquired drug resistance in tumor cells with more aggressive phenotype and angiogenic potential (for instance, pancreatic cancer and inflammatory breast cancer since they have similar molecular signatures).

Unpublished observations

Interactions between the EGF and cytokine receptor signaling networks

We have shown that combined stimulation of breast cancer cells with EGF and PRL or pancreatic cancer cells with EGF and Interleukin-6 (IL-6), which may act as pro-inflammatory or anti-inflammatory cytokine results in a synergistic induction of c-FOS mRNA and protein, which may be important for cell-fate decisions. We were investigating the mechanisms underlying this synergy phenomenon and building the mathematical model of ERK1/2 and STAT3-dependent signaling and regulation of c-Fos promoter region.

Recent (ongoing) research

Impact of voluntary cardiovascular exercise on single-pedicled fasciocutaneous flap survival in aged rat model

Fasciocutaneous flaps comprising skin, fat and deep fascia have been successfully used to cover a variety of traumatic and excisional defects in organs of head and neck, trunk as well as in upper or lower extremities. A disruption of local blood vessels during single-pedicled flap raising procedure causes an increase in cellular metabolism and oxygen consumption throughout the flap, producing a subcutaneous wound with reduced oxygen availability. Ischemic skin flap animal models were developed to simulate the effects of abnormalities present in human chronic wounds. These models are extensively used to examine the rate and properties of wound healing in response to physical (temperature, hypoxia, pressure etc.), endogenous (obesity, anemia, sex hormones, ageing etc.) and exogenous factors (e.g. treatment with natural or synthetic agents, exposure to radiation, gene therapy or physical challenge regimen). While bipedicle fasciocutaneous skin flap rat model produces moderate transient ischemia that causes delayed healing of full-thickness skin excision wounds, the rotational single-pedicle model produces more severe tissue ischemia that eventually leads to necrosis of the distal segment of the flap. We chose the latter model to investigate the effects of voluntary cardiovascular exercise on the ischemic flap survival in aged rats, since geriatric wound repair is known to be delayed and more prone to complications, such as infection, excessive scarring and development of pressure ulcers, whereas exercise reportedly accelerates cutaneous wound healing by suppressing inflammation in aged humans and rodents. We examined the expression kinetics of different apoptosis-related protein markers and cytokines in three different segments of the flap: proximal (the closest to the pedicle), middle and distal (this region is the most susceptible to necrosis) to correlate their expression profiles with flap necrosis percentage of the flap.

Single-pedicled fasciocutaneous flap survival in aged rat model of chronic alcoholism

In this experimental animal study, we investigate the effect of chronic alcohol intake on wound healing and survival of the single-pedicled rotational flap, which is based on inferior epigastric artery. We also examined the expression profile of different apoptosis- and fibrosis-related protein markers as well as growth factors and cytokines (e.g. VEGF, FGF-2, IL-6) in three different segments of the flap: proximal (the closest to the pedicle), middle and distal (this region is the most susceptible to necrosis) at 9th day post-surgery.

Future Research Goals

Currently we are developing the double flap animal model where we would be able to investigate the effects of different protein-targeted therapeutics on flap survival in various physiological settings (aged versus young rats, normal vs. alcoholic, lean vs. obese/diabetic, normal versus irradiated rats, etc.).