Somatostatin and Epidermal Growth Factor Receptors: Implications in Breast Cancer

Despite several advances, the underlying mechanism of complexity of breast cancer progression still remains elusive. In addition to the genetic predisposition, several growth factor receptors including insulin growth factor receptor (IGF), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) relaying proliferative signals are accountable for disease progression. Epidermal growth factor receptors (EGFRs, or commonly known as ErbBs), members of the receptor tyrosine kinase family (RTKs), play a central role in tumor growth, progression and metastatic disease. Typically, agonist dependent activation of EGFR results in receptor phosphorylation, homo- and/or heterodimerization and modulation of signaling pathways leading to cell proliferation, survival and metastasis. Targeting one or multiple steps in EGFR-mediated tumor progression may serve as a better approach in drug therapies. Unlike EGFRs, G-protein coupled somatostatin receptors (SSTRs) have been recognized as negative regulators of breast tumors. The activation of SSTRs modulates downstream signaling responsible for tumor growth and consequent cytostatic or cytotoxic effects on tumor proliferation. SSTR subtypes are well characterized to form homo-and/or heterodimers within the same family as well as with other GPCRs. Clinically, the chimeric molecule targeting both SSTR5 and dopamine receptors (specifically dopamine receptor 2) is in use for the treatment of pituitary tumors. This review describes the interplay between SSTRs and EGFR and the potential role of such cross talk in attenuation of EGFR-mediated signaling pathways involved in tumorigenesis. Furthermore, recent findings supporting the role of SSTR in EGFR-mediated signaling in tumor biology are discussed in detail

Somatostatin Receptor 1 and 5 Double Knockout Mice Mimic Neurochemical Changes of Huntington's Disease Transgenic Mice

Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD). However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST) positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5). and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease

Role of somatostatin receptors in epidermal growth factor mediated EGFR signaling in breast cancer and human embryonic kidney 293 cells

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.Pharmaceutical Sciences, Faculty ofGraduat

Inhibition of tumor promoting signals by activation of SSTR2 and opioid receptors in human breast cancer cells

Background: Somatostatin receptors (SSTRs) and opioid receptors (ORs) belong to the superfamily of G-protein coupled receptors and function as negative regulators of cell proliferation in breast cancer. In the present study, we determined the changes in SSTR subtype 2 (SSTR2) and μ, δ and κ-ORs expression, signaling cascades and apoptosis in three different breast cancer cells namely MCF-7, MDA-MB231 and T47D. Methods Immunocytochemistry and western blot analysis were employed to study the colocalization and changes in MAPKs (ERK1/2 and p38), cell survival pathway (PI3K/AKT) and tumor suppressor proteins (PTEN and p53) in breast cancer cell lines. The nature of cell death upon activation of SSTR2 or OR was analysed using flow cytometry analysis. Results The activation of SSTR2 and ORs modulate MAPKs (ERK1/2 and p38) in cell dependent and possibly estrogen receptor (ER) dependent manner. The activation of tumor suppressor proteins phosphatase and tensin homolog (PTEN) and p53 antagonized the PI3K/AKT cell survival pathway. Flow cytometry analyses reveal increased necrosis as opposed to apoptosis in MCF-7 and T47D cells when compared to ER negative MDA-MB231 cells. Furthermore, receptor and agonist dependent expression of ORs in SSTR2 immunoprecipitate suggest that SSTR2 and ORs might interact as heterodimers and inhibit epidermal growth factor receptor phosphorylation. Conclusion Taken together, findings indicate a new role for SSTR2/ORs in modulation of signaling pathways involved in cancer progression and provide novel therapeutic approaches in breast cancer treatment.Pharmaceutical Sciences, Faculty ofReviewedFacult

Correction: Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

<p>Correction: Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes</p

Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

<div><p>Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight^™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.</p></div

TEM analysis show presence of exosomes.

<p>Representative TEM images of exosomes derived from <b>a)</b> C42 PCa cell line <b>b)</b> LNCaP xenograft serum and <b>c)</b> patient plasma by ultracentrifugation method. Exosomes were negatively stained with 2% uracyl acetate after removal of moisture. Arrows indicate cup-shaped structures which are identified as exosomes (30–100 nm in diameter).</p

Exosome isolation from plasma is validated by the presence of exosome markers.

<p><b>a)</b> CD9 was present in exosomes derived from LNCaP xenograft mice bearing small, medium and large LNCaP tumours whereas the control mouse serum lacked CD9. GRP94, a known endoplasmic reticulum protein which is used as a negative control was absent in the exosomes suggesting enrichment <b>b)</b> LAMP2 was present in exosomes derived from PCa patient plasma whereas absence of LAMP2 in whole plasma indicated successful enrichment. <b>c)</b> Alix was present in exosomes derived from patient plasma at different exosomal protein concentrations.</p

Representative graphs of NanoSight^™ particle tracking analysis.

<p>The analysis showed that mean size of exosomes isolated from control mouse was 126 nm <b>(a)</b> whereas LNCaP xenografted mice bearing small tumour was 81nm (b), 137 nm from medium <b>(c)</b> and 67 nm from large tumours <b>(d)</b>. The concentration of exosomes secreted increased with the increasing size of the tumour.</p

Information of PCa plasma and serum used to derive exosomes.

<p>Information of PCa plasma and serum used to derive exosomes.</p