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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
Somatostatin (SST) inhibits cell proliferation through five SST receptors(SSTR1-5). Amongst all SSTR subtypes, SSTR2 and SSTR3 are the prominentreceptor subtypes which exert antiproliferative effects in cells of different origin.SSTR2-mediated inhibition of cell proliferation is largely cytostatic, whereas SSTR3is cytotoxic. Whether SSTR2/SSTR3 display synergistic antiproliferation than singlereceptor is not well understood. To ascertain the role of SSTR3, the present studywas first conducted in HEK-293 cells which lack endogenous SSTRs expression.Cells were stably transfected with wt-SSTR3, treated with agonist and studied fordimerization, cAMP, receptor trafficking and signaling molecules. Since receptorsignaling properties are confined in C-tail, cells expressing C-tail deleted SSTR3were also studied for comparative analysis. wt-SSTR3 exists as preformedhomodimer at cell surface and displays agonist-mediated cytotoxic effects. The cellsurface expression, homodimerization and agonist-induced internalization of SSTR3were independent of C-tail, whereas agonist-mediated apoptosis was lost upon Ctaildeletion.Next, HEK-293 cells cotransfected with SSTR2/SSTR3 were examined forheterodimerization and signaling molecules governing cell proliferation. Pb-FRET/CO-IP analyses suggest SSTR2/SSTR3 heterodimerization. The decreasedcAMP upon agonist activation of SSTR2/SSTR3 suggests that this heterodimer isfunctional. Agonist-mediated SSTR2/SSTR3 antiproliferation was Gi-dependent, andinvolved apoptosis and cell-cycle arrest. iiiTo derive direct pathological significance of the observations from heterologous system, additional experiments were conducted in two breast cancer cell lines MCF-7 and MDA-MB-231, which differ in origin and biochemical features including presence or absence of ERα. Breast tumor cell lines overexpressing SSTR3 were studied for cell proliferation and downstream signaling molecules. EGF served as an index of positive cell proliferation. SSTR3 overexpression in MCF-7 (R3-MCF-7) and MDA-MB-231 (R3-MB-231) cells displayed inhibition of EGF-induced proliferation and enhanced antiproliferative effect of SSTR3-specific agonist in comparison to non-transfected cells. SSTR3 overexpression in R3-MCF-7 cells constitutively enhanced TUNEL staining, PARP-1 and p27Kip1 expression suggesting apoptosis and cell-cycle arrest. Conversely, in R3-MB-231 cells, SSTR3 overexpression exerted cytostatic but not cytotoxic effects.These results provide compelling evidence for antiproliferative role of SSTR3 in breast cancer cell lines. The constitutive activation of cytotoxic signaling in R3-MCF-7 but not R3-MB-231 cells reveals a distinct cell-specific role for SSTR3 in breast tumor biology.
Somatostatin (SST) is a multifunctional peptide present in most brain regions as well as inperipheral organs. In QUIN/NMDA-induced excitotoxicity, an experimental model ofHuntington’s disease (HD), SST positive interneuron coexpressing NPY/NADPH-d/bNOSare selectively spared whereas, projection neurons expressing NMDA receptors and DARPP-32 are vulnerable. SST plays neuroprotective role in excitotoxicity, however, which SSTRsubtypes mediate the neuroprotective role is not known. Accordingly, as a first step, wedescribe the colocaliztion of SSTR subtypes with DARPP-32 to determine the percentage ofreceptor subtypes in projection neurons. We further extended our study and compared HDtransgenic mice (R6/2) with SSTR1 and 5 double knock out mice. In both strains wecompared the expression pattern of NMDARs, DARPP-32, SST, bNOS and SSTRs and keydownstream signaling pathways linked to the neuronal loss in HD such as PI3K, ERK1/2PKC-α, synapsin-IIa, enkephalin and calpain. Our data shows that SSTR1/5 double knockout mice mimic the neurochemical changes of HD transgenic mice indicating a keyneuroprotective role of SSTR1 and 5 in HD. To derive direct physiological implications andmechanistic explanations for the role of SSTR subtypes in excitotoxicity we used striatalbrain slices and determined the effect of SSTR1 and 5 agonist, alone or in combination withNMDA on key proteins such as DARPP-32, calpain, PSD-95 and signaling pathwaysassociated with NMDA induced neurotoxicity. Our results here show significant decrease inNMDA currents and dissociation of NMDARs heterodimerization upon treatment withSSTR1 and 5-specific agonist. Our data further demonstrates significant decrease inNMDARs expression and upregulation of SSTR1 and 5 upon agonist treatment. UnlikeNMDA, activation of SSTR1 or 5 in striatal slices induced DARPP-32 phosphorylation atThr34 and Thr75 enhanced CREB phosphorylation and inhibits expression of calpain andPSD-95. The data presented in this thesis provides a new insight for the role of SSTRsubtypes in excitotoxicity with relevance to neurological disorders.
Epidermal growth factor receptor (ErbB1) and somatostatin receptors (SSTRs) exert opposing effects on tumor promoting signaling pathways. Whether SSTRs functionally interact with ErbB1 and modulate tumor-promoting signaling is currently unknown. For this reason, the specific emphasis of this thesis is to examine the role of SSTRs in ErbB1 mediated signaling in breast cancer cells and human embryonic kidney (HEK) 293 cells. First, I determined the mRNA and protein expression of SSTR1, SSTR5 and ErbB1 in human breast cancer cell lines namely MCF-7 and MDA-MB231. I next demonstrated that SSTR1 or 5 exist as pre-formed heterodimers with ErbB1, which dissociated in an agonist dependent manner. Somatostatin (SST) modulated epidermal growth factor (EGF) mediated MAPK in a time and agonist dependent manner. Furthermore, SST and/or EGF treatment altered the expression of key adapter proteins including Grb2, SOS, Shc, SH-PTP1 and SH-PTP2, which are known to play a role in MAPK activation. Since breast cancer cells endogenously express SSTR and ErbB subtypes, this study was further extended in HEK-293 to gain insight into the effect of individual SSTR on ErbB1 activated signaling. We demonstrated that HEK-293 cells transfected with SSTR1, SSTR5 or SSTR1/5 negatively regulate EGF mediated effects attributed to the inhibition of ErbB1 phosphorylation, MAPKs and PI3K/AKT pathways. Moreover, SST effects were significantly enhanced in cells when ErbB1 was knocked down using small interference ribonucleic acid (siRNA) or treated with selective ErbB1 antagonist (AG1478). The presence of SSTRs, in addition to modulating signaling pathways, led to the dissociation of constitutive and EGF induced heteromeric complex of ErbB1/ErbB2. Most significantly, cells co-transfected with SSTR1/5 display pronounced effects of SST on the signaling and dissociation of the ErbB1/ErbB2 heteromeric complex than cells expressing either SSTR1 or 5 alone. The findings of this study discovered a new mechanism and potential role of SSTRs in attenuation of ErbB1 mediated signaling pathways via dissociation of ErbB1/ErbB2 heteromeric complex. In conclusion, the results presented in this thesis suggest that formulating novel drugs that activate SSTRs along with inhibition of ErbB1 might likely serve as an important therapeutic approach in the treatment of ErbBs positive tumors.
Master's Student Supervision (2010 - 2018)
The molecular mechanisms of breast cancer are poorly understood, which present serious therapeutic problems and complicates drug design. Cell surface receptors belonging to G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) families are potential drug targets relevant to pathological conditions, and have attracted great interest from pharmaceutical industry. Recent studies have suggested that somatostatin (SST) receptors (SSTR1-5) belonging to GPCR family may interact with human epidermal growth factor (EGF) receptors (ErbB 1-4) from RTK family in pathophysiological conditions, exerting antiproliferative effects that may be useful in the treatment of breast cancers. An understanding of molecular mechanisms responsible for these effects reveal new approaches to the design of efficient breast cancer therapies that would significantly improve the lives of patients. The work presented in this thesis was conducted to investigate crosstalk between SSTR and ErbB proteins in BT-474 and SK-BR-3 breast cancer cell lines upon SST and/or EGF treatment, to clarify the underlying molecular mechanisms, and to explore their implications for cancer therapy. Several pairs of SSTR and ErbB proteins exhibited strong membrane coexpression and crosstalk in the presence of the tested ligands, including SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 in BT-474 cells, and SSTR5/ErbB1 in SK-BR-3 cells. New crosstalk processes between SSTR and ErbB subtypes were observed in both cell lines. In BT-474 cells, there were substantial reductions in the membrane expression of ErbB1 (degradation and termination of signaling) and ErbB2, as well as moderately reduced expression of ErbB3 and greatly enhanced activation of SSTR1 and SSTR4. Similarly, SK-BR-3 cells exhibited strong reductions in the expression of ErbB1 (degradation), ErbB3, and ErbB2 expression (partial degradation), while enhanced activation of SSTR1 and SSTR2 expression at the cell surface. The activated SSTRs were shown to antagonize ErbB-mediated MAPK signaling and tumor-promoting signaling pathways, resulting in pronounced antiproliferative effects. In BT-474 cells, they inhibited ERK1/2, p38 and PI3K, and enhanced PTEN pathways, while in SK-BR-3 cells they promoted ERK1/2 and p38, inhibited PI3K and maintained PTEN pathways. These results show that the activated SSTRs exert antiproliferative effects in breast cancer cells via mechanisms that resemble those determined for drugs modulating cancer-related signaling pathways.
NMDA receptors are glutamate-gated cation channels named after their prototypical selective agonist NMDA. The channels occur as multiple subtypes, which are formed from interactions between different receptor subunits. NMDA receptor subunits are classified into three families: NR1, NR2A-D, and NR3A, B. NMDA receptors are implicated in HD pathology. During HD, a subset of medium-sized aspiny interneurons in the striatum that co-localize SST, NPY, and the enzyme NOS are selectively spared. In contrast, medium-sized spiny cells that constitute 80 % of all striatal neurons undergo selective neurodegeneration. While it was suggested that the interneurons survive because they lack NMDA receptors, studies including from our lab have shown the presence of NR1 in SST-positive striatal neurons. The finding of NR1 expression and co-localization with SST-positive neurons indicates that NMDA receptor-induced toxicity may be regulated in a receptor-specific manner. Therefore, the present study was conducted to investigate whether NMDA application leads to toxicity that is receptor-specific in HEK293 cells stably transfected with NR1, NR2A, or NR2B. The main findings of this study indicate that NMDA application causes cell death, which varies in intensity and nature, depending upon the NMDA concentration applied, and the receptor-type expressed by the cells. Cells expressing NR1 were found to undergo apoptosis but not necrosis, while cells expressing NR2A/NR2B underwent both apoptosis and necrosis in a receptor-specific manner. In cells expressing NR2A/NR2B, exposure to low concentrations of NMDA resulted in cell death that was predominantly apoptotic. In contrast, exposure to high concentrations of NMDA produced mostly necrosis. In cells expressing NR1, NMDA application caused apoptosis, which exhibited a gradual increase in response to greater concentrations of NMDA. In addition, cell death through apoptosis and/or necrosis was determined to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. Taken together, these results indicate that the activation of receptors formed by NR1, NR2A, or NR2B have different toxic consequences. Thus, the selective neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons.
No abstract available.