Aug
12
Mon
2013
Delegate Talk: AIB1 Mediated Modulation of CXCR4-SDF1 Signaling in Breast Cancer @ Acharya Hall
Aug 12 @ 3:23 pm – 3:34 pm
Delegate Talk:  AIB1 Mediated Modulation of CXCR4-SDF1 Signaling in Breast Cancer @ Acharya Hall | Vallikavu | Kerala | India

Binu K Aa, Jem Prabhakarb, Thara Sc and Lakshmi Sd,

aDepartment of Clinical Diagnostics Services and Translational Research, Malabar Cancer Centre, Thalassery, Kerala, India.
bDivision of Surgical Oncology, Division of Pathology
dDivision of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India.


Introduction

AIB1, a member of the nuclear co activators, promotes the transcriptional activity of multiple nuclear receptors such as the ER and other transcription factors. Chemokines produced by stromal cells have potential to influence ERα-positive breast cancer progression to metastasis. CXCR4 is the physiological receptor for SDF1, together shown to stimulate the chemotactic and invasive behavior of breast cancer cells to serve as a homing mechanism to sites of metastasis. We propose that over expression of AIB1 in breast cancer cells leads to increased SDF1 and CXCR4 expression, which induces invasion and metastasis of cancer cells.

Materials and Methods
Breast tumor and normal breast tissues from patients in Regional Cancer Centre, Thiruvananthapuram were used for study. The modulatory effect of AIB1 was studied in MCF-7 cells with AIB1 siRNA transfection along with treatment of 17β-Estradiol (E2), 4-hydroxytamoxifen (4OHT), combinations of E2 and 4OHT. The gene expression pattern and protein localization were assessed by RT-PCR and immunofluorescence microscopy respectively. The metastatic and invasive properties were assessed by wound healing assay. Quantitative colocalization analyses were done to assess the association of proteins using Pearson’s correlation coefficient.

Result and Conclusion
The mRNA and protein level expression of AIB1, CXCR4 and SDF1 were higher in tumor samples than in normal samples. AIB1 was localized to the nuclei whereas CXCR4 and SDF1 immunoreactivity were observed in the cytoplasm and to a lesser extent in the nuclei of tumor epithelial cells. In tumor samples the gene level expressions of AIB1 showed significant positive correlations with SDF1(r = 0.213, p = 0.018). CXCR4 showed significant positive correlation with SDF1 in gene (r = 0.498, p = 0.000) and protein levels(r = 0.375, p = 0.002). Quantitative colocalization analyses showed a marked reduction in expression of CXCR4 and SDF1 in siAIB1MCF-7 cells than MCF-7 cells with different treatment groups. Wound healing assay shows reduced wound healing in siAIB1 treated MCF-7 cells.

In recent years, targeting specific cancer pathways and key molecules to arrest tumor growth and achieve tumor eradication have proven a challenge; due to acquired resistance and homing of cancer cells to various metastatic sites. The present study revealed that silencing AIB1 can prevent the over expression of SDF1 and CXCR4. Co activator levels determine the basal and estrogen-inducible expression of SDF1, a secreted protein that controls breast cancer cell proliferation and invasion through autocrine and paracrine mechanisms (Hall et al. 2003). The effects of CXCR4 overexpression has been correlated with SDF1 mediated activation of downstream signaling via ERK1/2 and p38 MAPK and with an enhancement of ER-mediated gene expression (Rhodes et al. 2011). It is possible that over expression of AIB1 as a stimulant involved in the expression of CXCR4 might up-regulate the expression of prometastatic and angiogenic genes. Thus based on these observations it can be concluded that SDF1/CXCR4 overexpression, with significant association with AIB1 expression, itself contribute to the development of mammary cancer and metastatic progression.

Aug
13
Tue
2013
Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall
Aug 13 @ 6:18 pm – 6:25 pm
Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall | Vallikavu | Kerala | India

Manjunath Joshi, Apoorva Lad, Bharat Prasad Alevoor, Aswath Balakrishnan, Lingadakai Ramachandra and Kapaettu Satyamoorthy


 

Pathological conditions during Type 2 Diabetes (T2D) are associated with elevated risk for common community acquired infections due to poor glycemic control. Multiple studies have indicated specific defects in innate and adaptive immune function in diabetic subjects. Neutrophils play an important role in eliminating pathogens as an active constituent of innate immune system. Apart from canonically known phagocytosis mechanism, neutrophils are endowed with a unique ability to produce extracellular traps (NETs) to kill pathogens by expelling DNA coated with bactericidal proteins and histone. NETosis is stimulated by diverse bacteria and their products, fungi, protozoans, cytokines, phorbol esters and by activated platelets. Considering deregulation of metabolic and immune response pathways during pathological state of diabetes and NETosis as a potential mechanism for killing bacteria, we therefore, investigated whether hyperglycemic conditions modulate formation of neutrophil NETs and attempted to identify underlying immunoregulatory mechanisms. Freshly isolated neutrophils from normal individuals were cultured in absence or presence of high glucose (different concentrations) for 24 hours and activated with either LPS (2 mg/ml) or PMA (20 ng/ml) or IL-6 (20 ng/ml) for 3 hours. NETs were visualized and quantified by addition of DNA binding dye SYTOX green using fluorescence microscope and fluorimetry. NETs were quantified in Normal and diabetic subjects. Serum IL-6 levels were measured using ELISA technique. NETs bound elasatse were quantified in normal and diabetic subjects in presence or absence of DNase. Bacterial killing assays were performed upon infecting E.coli with activated neutrophils from normal and diabetic subjects. Microscopy and fluorimetry analysis suggested dramatic impairment in NETs formation under high glucose conditions. Extracellular DNA lattices formed in hyperglycemic conditions were short lived and unstable leading to rapid disintegration. Subsequent, time course experiments showed that NETs production was delayed in hyperglycemic conditions. To validate our findings more closely to clinical conditions, we investigated the neutrophil activation and NETs formation in diabetic patients. Upon stimulation with LPS for three hours, neutrophils from diabetic subjects responded weakly to LPS and lesser NETs were formed; whereas, neutrophils from normal individuals showed robust release of NETs. In few patients we found short and imperfect NETs in basal conditions suggesting constitutive activation of neutrophils in diabetic subjects. Interestingly, NETs bound elastase activity was reduced in diabetes subjects when compared to non-diabetic individuals, indicating a dysfunction of one of the important protein component of NETs during diabetes. Neutrophils from diabetic subjects released higher levels of IL-6 without any stimulation suggesting an existence of constitutively activated pro-inflammatory state. IL-6 induced NETs formation and was abrogated by high glucose. Weobserved that glycolysis inhibitor 2-DG resensitize the high glucose attenuated LPS and IL-6 induced NETs. a) NETs are influenced by glucose homeostasis, b) IL-6 as potent inducer of energy dependent NETs formation and c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible for infections.