Proteomic Profiling of Î²-hCG-Induced Spheres in BRCA1 Defective

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Proteomic profiling of #-hCG induced spheres in BRCA1 defective triple negative breast cancer cells Satheesh Kumar Sengodan, Arathi Rajan, Sreelatha Krishnakumar Hemalatha, Revathy Nadhan, Abdul Jaleel, and Priya Srinivas J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00562 • Publication Date (Web): 13 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Journal of Proteome Research

Proteomic profiling of β-hCG induced spheres in BRCA1 defective triple negative breast cancer cells Satheesh Kumar Sengodan1, Arathi Rajan1, Sreelatha Krishnakumar Hemalatha1, Revathy Nadhan1, Abdul Jaleel2, Priya Srinivas* 1

Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram,

Kerala, India. 2Cardiovascular Diseases and Diabetes Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India. *Correspondence to: Dr. Priya Srinivas, Cancer Research, Rajiv Gandhi Centre for Biotechnology, Thycaud PO, Thiruvananthapuram 695 014, Kerala, India. Tel: 91-471- 234 7975; Fax: 91- 471- 23 48 096. Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract Previously we identified that β-hCG is expressed by BRCA1 mutated but not wild type breast cancers in-vitro/in-vivo and exhibit a novel event in β-hCG over expressing BRCA1 mutated HCC1937cells where the cells were able to form spheres in adherent cell culture plates even in the absence of any growth factors. These spheres express stem cell and EMT markers. In the present study we carried out the total proteomic profiling of these HCC1937 β spheres obtained from BRCA1 defective β-hCG expressing stable breast cancer cells to analyze the cell signaling pathways that are active in these cells. Functional annotation revealed proteins (164 cellular and 97 secretory) predominantly involved in oxygen binding, nucleosome assembly, cytoskeleton organization, protein folding etc. Many of the proteins identified from HCC1937 β spheres in this study are also up regulated in breast cancers which are directly linked with poor prognosis in human cancer samples as analyzed using TCGA dataset. Survival analysis shows that β-hCG expressing cancer patients are linked with poor survival rate. Interestingly, haemoglobins were identified at both cellular and secretary level in HCC1937 β spheres and experiments after treating with ROS inducers revealed that β-hCG induces haemoglobin and protects the cancer cells during oxidative stress. Our proteomic data strongly propose β-hCG as an oncogenic molecule associated with BRCA1 mutation and hence targeting β-hCG could be a strategy to treat BRCA1 defective breast cancers. Keywords: HCC1937 β Spheres, BRCA1, Proteomics, Haemoglobin, TCGA, Secretory proteins, Cellular proteins, Mass spectrometry, Breast cancer


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Introduction Presence of Cancer Stem Cells (CSCs) has been widely accepted among cancer researchers and drug resistance by CSCs remains a challenge even after several years of research. CSCs are a discrete population of cancer cells which possess the property of self renewal and differentiation. They are highly tumorigenic and have been identified by expression of several markers such as OCT4, Nanog, SOX2 etc (1). Sphere formation is the key phenomenon which represents the stem cell nature of cancer cells in-vitro. Loss or mutation of certain tumor suppressor genes has been identified to induce stem cell nature of cancer cells (2). For instance, BRCA1 defectiveness has been reported to exhibit high stemness in breast cancer (3, 4), but the molecular pathways are not completely understood. Though there are many oncogenic factors reported in BRCA1 defective cancers, recently we have shown β-hCG, a pregnancy hormone to be involved in inducing tumorigenesis of BRCA1 defective cancers (5). Mammosphere cultures are done to analyze breast cancer stem cell characteristics using non-adherent cell culture plates with stem cell media which is rich in growth factors. We have identified for the first time, the induction of sphere formation by β-hCG over expressing BRCA1 mutant breast cancer cells in-vitro in adherent cell culture plates even without supplementation of any growth factors (5). Further these HCC1937 β spheres were found to express high levels of CSCs and EMT markers. Therefore, in this study we carried out the total proteomic profiling (cellular and secretory proteins) of HCC1937 β spheres obtained from BRCA1 defective β-hCG expressing stable breast cancer cells which has not been analyzed yet.

Materials and Methods



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Cell culture HCC1937 and HCC1937/wt BRCA1 cell lines were a kind gift from Dr. Grant Mc Arthur, Peter MacCallum Cancer Centre, VIC, Australia. Stable over expression of β-hCG plasmid (16574, Addgene) in HCC1937 resulted in the formation of spheres (HCC1937 β spheres) in adherent cell culture plates. The spheres were collected by centrifuging the supernatant at 1000g for 10 min. For proteomic analysis, the spheres was washed with 1X PBS and seeded in serum free RPM1-1640 media and maintained at 370C in CO2 incubator for 5 days. Sample preparation for LC/MS For cellular proteins, the HCC1937 β spheres, HCC1937 adherent cells and HCC1937/wt BRCA1 adherent cells were washed with 1X PBS for 5 min by centrifugation at 1000 rpm and 200 mM rapigest (Waters, 186001860) dissolved in ABC buffer (50 mM Ammonium bicarbonate +200 mM PMSF+ benzonase nuclease) was used for protein isolation. For secretory proteins, the conditioned supernatant medium from HCC1937 β spheres was concentrated using Amicon 3kDa filter (Amicon, UFC800324) and buffer exchanged with 50 mM ammonium bicarbonate buffer. Both cellular and secretory proteins were quantified using Bradford reagent (Biorad, 500-0006) and a concentration of 1ug/ul proteins were used for LC/MS analysis. The disulphide bonds of the protein samples were reduced using 10 mM DL-dithiothreitol at 600C for 30 min and were alkylated using 200 mM iodoacetamide at 270C in the dark for 20 min. Insolution trypsin digestion of samples was carried using sequencing-grade modified trypsin at 370C overnight. Trypsin digestion was stopped by adding formic acid and the digested peptides were centrifuged at 40C for 12 min at 13,200 rpm and the supernatant was stored at -800C until use.


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Liquid Chromatography, Mass spectrometry, Protein identification and relative protein quantitation Nano-LC/MSE (MS at elevated energy) was used to analyze the peptide samples using a nano ACQUITY UPLC® System (Waters, UK) coupled to a Quadrupole-Time of Flight (Q/TOF) mass spectrometer (SYNAPT-G2, Waters, UK) where the peptides were separated by reverse phase column chromatography. MassLynx4.1 SCN781 software (Waters, UK) was used to operate both the systems. 5μl of samples was injected in partial loop mode and the complete chromatographic protocol was carried out as described previously (6). The temperature of the column was maintained at 400C. SYNAPT G2 High Definition Mass Spectrometer — HDMS (Waters) was used for analyzing the eluting peptides from LC. The parameters and the calibration of the Mass Spectrometer were chosen as described earlier (6). ProteinLynx Global Server version 2.5.3 (PLGS, Waters) was used to analyze the LC–MSE data for protein identification and relative quantitation. Database search was done using Human database downloaded from NCBI and the protein false positive rate was set at 4%. The data was processed, analyzed, normalized as described earlier (6). For relative quantitation of HCC1937 and HCC1937/wt BRCA1, auto-normalization function of PLGS was used to normalize the datasets and quantitative analysis was done by comparing the normalized peak area/intensity of the peptides. ADH was used as an internal control for normalization. Thus, number of peptides and sequence coverage for each protein were obtained and the proteins were considered to be significantly altered if p-value >0.95 (up regulated) and p-value

Proteomic Profiling of Î²-hCG-Induced Spheres in BRCA1 Defective

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