Download The Role of MET in the Proliferation of Papillary Renal...

The Role of MET in the Proliferation of Papillary Renal Cell Carcinoma
Kylee Rosette, Stephen Lander, Calvin Van Opstall, and Brendan Looyenga, PhD
Department of Chemistry & Biochemistry, Calvin College, Grand Rapids, Michigan
Introduction
Papillary renal cell carcinoma (RCC) is
the second most common type of kidney
cancer (1 in 10 renal cell carcinomas).
MET:
MET, a membrane receptor protein, is found to
be over-expressed in these particular types of
tumors.
Figure 1: MET is a
dimerized protein
located in the cell
membrane. It binds
to hepatocyte growth
factor (HGF) to
stimulate various
proliferation
pathways. (adapted
Objectives
Results
Conclusions
Determine if the pharmacologic
MET inhibitor (INCB28062) results
in the same decrease in MET activity
as shRNA knockdown of MET
Validation of MET shRNA Chronic
Knockdown:
Chronic knockdown of MET
expression (shRNA) is effective in limiting
proliferation in two dimensions
Determine under what conditions, if
any, the MET inhibitor will decrease
cell proliferation
Methods
Types of Cells Used:
from Janku et al).
The Big Question:
After inhibiting the function of MET in two
different ways (RNA interference vs.
pharmacologic inhibition), it was found that
only knockdown actually slowed cell
proliferation. This is surprising because both
methods should inhibit MET function to a
similar degree.
Figure 4: Comparing the amount of active MET (pMET) to
the amount of total MET (MET) in different shRNA
knockdown conditions (M1, M2, M3, M4). A control serves
as the standard of comparison (NT). The SKRC39 cell line,
not shown, presents similar results to the Caki2 cell line.
Actin serves as a loading standard
Validation of INCB MET Acute
Inhibition:
Acute inhibition of MET activity
(INCB028060) may be effective in
limiting proliferation in 3D
Future Directions
 HK2: Normal human kidney cell line
Replicate results obtained from the soft
agar assay
SKRC39: Malignant Papillary RCC line
 Caki2: Malignant Papillary RCC line
Find additional methods for testing
INCB effectiveness in 3D growth
Western Blotting:
Figure 5: Comparing the amount of active MET (pMET) in
different INCB concentration conditions. Tested in SKRC39
cells grown in FBS media. This shows the effective
inhibition of active MET even at low INCB concentrations
A)
shRNA Chronic MET
Expression
Knockdown:
Test the effectiveness of INCB in low
nutrient media conditions
References
inhibited cellular
proliferation
INCB028060 Acute
MET Activity
Inhibition:
Acute inhibition of MET activity
(INCB028060) is not effective in limiting
proliferation in two dimensions
Table 1: Various conditions examining the
effect of INCB on cell proliferation
B)
Condition
Varying
Doses of
Drug
did not inhibit
cellular
proliferation
Figure 2: A real-time measurement (Xcelligence) of cell
proliferation in malignant Caki2 cells. Condition A)
removes the genomic expression of MET from the DNA
of the cell so that it can no longer produce any active
MET . Condition B) uses the drug inhibitor
INCB028060 to stop the activity of the MET protein.
The control for both conditions is shown in blue.
Figure 3: Lysed cell proteins are run on gel
electrophoresis, then washed in primary and
secondary antibodies. The secondary antibody has
fluorescent properties, in which higher fluorescence
correlates to greater amounts of protein from the
cell. The darker the band, the greater amount of
protein present
Testing
Impact of drug Effective knockdown of
concentration MET and no affect on
on growth
proliferation
Immediately
Impact on
after Plating initiating cell
Cells
cycle
Soft Agar
Assay
Outcome
Effective knockdown of
MET and no affect on
proliferation
Preliminary results show
Impact on 3D reduced growth in INCB
growth
condition compared to
control
Funding for this project was provided by the National Cancer
Institute (R15 CA192094-01)
Looyenga B, et al. Chromosomal amplification of leucine-rich
repeat kinase-2 (LRRK2) is required for oncogenic MET
signaling in papillary renal and thyroid carcinomas
Proceedings of the National Academy of Science. 25 Jan
2011. 108(4): 1439-44. PMID: 21220347.
Ding Y, et al. Combined gene expression profiling and RNAi
screening in clear cell renal cell carcinoma identify PLK1
and other therapeutic kinase targets. Cancer Research. 1
Aug 2011. 71(15):5225-34. PMID: 21642374.
Albiges L, et al. MET is a potential target across all papillary
renal cell carcinomas. Clinical Cancer Research. 1 July
2014. 20(13):3411-21. PMID: 24658158
Filip Janku, David J. Stewart & Razelle Kurzrock ,Nature
Reviews Clinical Oncology 7, 401-414 (July 2010)