1. Analysis of eHsp90 Regulation of Secreted Exosomes
from Tumor Cancer Cells
Juan F.Toscano, Daniel S. Wong, Daniel G.Jay
Building Diversity in Biomedical Sciences Program (BDBS), Department of Natural Sciense, Assumption College,
Sackler School of Graduate Biomedical Science , Tufts University School of Medicine, Boston MA 0211
Background
• Cancer is most deadly at metastatic stage of the disease.
Under stress, cancer cells release a high amount of micro-
vesicles named exosomes.
• They have a high number of number of
proteins and micro-RNA (miRNA) that
could enhance cancer invasiveness
and metastasis.
• Exosomes could be a way for distant cells to communicate
and exchange genetic information outside the cell.
• The chaperone protein heat shock
protein 90 (Hsp90) is an abundant
intracellular protein. It is
secreted from the cell in exosomes
and activates pro-invasive proteins
that contribute to cellular migration.
Experimental Design
• Breast cancer cells (MDA-MB-231) media was treated with
two different Hsp90 inhibitor drugs at different
concentrations, and exosome isolated.
• This is followed by a BCA protein assay to quantify the
protein concentration present in the exosomes.
• Based on the protein concentration obtained, samples are
subjected to a SDS PAGE followed by a western blot
probed with anti Hsp90 antibody.
Conclusions and Future Experiments
MDA-MB-231
Untreated +100nm STA-12-7191
+ DMSO +10nm STA-12-7191
Exosome Isolation +10nm ganetespib
Exosomes Exosomes Exosomes Exosomes Exosomes
BCA Protein Assay
SDS PAGE/Western blot
• We will correlate Hsp90 activity with the number of
exosomes that can be isolated from the extracellular space
of breast cancer cell line MDA-MB-231
• If the exosome release from cancer cells decreases by
inhibiting eHsp90 with drugs, we will know that Hsp90
regulates the exosomal release process.
Results
• Hsp90 was found in exosomes released from breast cancer
cells.
• Cells that were treated with hsp90 inhibitors showed a
decrease exosomal protein concentration.
• Western blots probed with hsp90 antibody are required to
support the hypothesis that ganetespib and STA-12-7191
decreases exosome release from breast cancer cells.
• Once I leave Tufts, I will continue working on this project at
Assumption College during the school year.
• If we obtain promising data, we can isolate exosomes from
serum from cancer patients and probe for Hsp90.
• We can also see if Hsp90 is important for trafficking
exosomes to other cells by fluorescently tagged proteins
and/or miRNA.
Control Treatment
• The BCA Assay showed protein concentration present outside
the cell (Media) in exosomes.
• The first western blot support the presence of Hsp90 outside
cancer cells.
• Based on the darkness of the bands, and how much sample we
loaded to the gel (3µg/well), we observed that Hsp90 was a very
concentrated protein in the extracellular space.
Acknowledgements and Reference
This research was supported by grant number NIH 5R25
HL007785-22. I would like to thank the Jay lab members and the
Building Diversity in Biomedical Science program for this wonderful
research experience this last ten weeks.
McCready, J.; Sims, J.D.; Chan, D.; Jay, D.G. Secretion of extracellular
hsp90alpha via exosomes increases cancer cell motility: A role for
plasminogen activation. BMC Cancer 2010, 10, 29
Azmi, Asfar S., Bin Bao, and Fazlul H. Sarkar. "Exosomes in Cancer
Development,Metastasis, and Drug Resistance: A Comprehensive
Review." Cancer and Metastasis Reviews 32.3-4 (2013): 623-42. Web.
McCready J.; Wong D.; Burlison J.A; Ying W.; Jay D.G. “An Impermeant
Ganetespib Analog Inhibits Extracellular Hsp90-Mediated Cancer Cell
Migration that Involves Lysyl Oxidase 2-likeProtein” Cancers 2014, 6,
1031-1046.
Hypothesis
• We hypothesize that Hsp90 regulates exosome secretion
from tumor cancer cells.
• If the exosome release from cancer cells decreases by
inhibiting eHsp90 with drugs, we will know that Hsp90
regulates the exosomal release process. Inhibition of
Hsp90 could be a means to prevent cancer metastasis
• We will test this hypothesis by monitoring exosome release
from cells in the presence of Hsp90 inhibitors.
Drug treatment
• BCA protein assay showed detectable levels of protein
concentration.
• The control samples showed similar lavels of exosomal protein
concentration.
• The media treated with drug showed a decrease in exosomal
protein concentration.