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1. Abstract
The investigation carried out in this paper consisted of a pair of experiments designed to
answer the question, “Does the degree of mesenchymal phenotype affect the sensitivity of non-
small cell lung cancer cell lines to TBK1 inhibitors?” The hypothesis formulated was that if
different variants of a cell line were modified to express mesenchymal or epithelial
characteristics and were then treated with TBK1 inhibitors, then the cells that have a more
mesenchymal phenotype will show a greater sensitivity to TBK1 inhibitors than cells with a
more epithelial phenotype. Two experiments were carried out to test the hypothesis: a 96-well
plate drug response curve was generated to test the drugs’ ability to stop cellular proliferation
and a 6-well binary assay was used to test the drugs’ ability to kill off the cells. The data from
the 96-well plate was normalized twice and put into graph format, whereas the 6-well binary
assay plates were photographed and compared to determine the effects of the drugs on the cell’s
confluency. The experiments supported the hypothesis: in both experiments, data supported the
hypothesis that cells with a more mesenchymal phenotype are more sensitive to TBK1 inhibition
than are cells with a more epithelial phenotype. Further testing could involve rerunning the 6-
well assay and quantifying the amount of cells in each well, running a Western Blot to see the
protein levels of mesenchymal and epithelial indicators in the cell lines, and running tumor
studies in mice to test the efficacy of the drugs in vivo. The results from these experiments
indicate that TBK1 inhibition could be a viable option for targeting late-stage cancers as well as
mutant Ras cancers, though further experimentation is necessary to test the prospects of TBK1
inhibition as a method of combating said cancers.
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2. Introduction
This experimental analysis seeks to look at kinase inhibition as a method of stopping
proliferation of and killing off cancer cells. This analysis utilized non-small cell lung cancer cell
lines and looked at the sensitivity of said cell lines to inhibition of TBK1, a kinase that is
involved in multiple cell processes and an integral downstream effector of the RalGEF pathway,
a downstream pathway of the oncogenic Ras protein (Cooper, et al., 2013) (Ou, et al., 2011). The
specific focus of this analysis is to determine the effect of a mesenchymal phenotype on the
sensitivity of non-small cell lung cancer cell (NSCLC) lines to TBK1 inhibitors. The question
this analysis seeks to answer is, “Does the degree of mesenchymal characteristics affect TBK1
sensitivity in non-small cell lung cancer cell lines?” The hypothesis before undertaking this
investigation is that if different variants of the same non-small cell lung cancer cell line are
treated with TBK1 inhibitors, then cell line variants displaying a more mesenchymal phenotype
will be more sensitive to TBK1 inhibition than variants of the same cell line that display a more
epithelial phenotype.
3. Background
Cancer is a prominent disease. It kills 8.2 million people every year and has 14 million
new cases every year (National Cancer Institute, 2015). Finding cures for the many different
types of cancer is a top research priority today. One of the main areas of research regarding
cancer treatment centers on the Ras oncogene and its mutated status. The Ras oncogene pathway
is part of signal transduction in cells, and it is involved in various cell processes, such as cell
cycle progression, growth, migration, apoptosis, and cell proliferation (Fernández-Medarde &
Santos, 2011). Cancers that have Ras mutations are some of the worst cancers that exist. Up to
30% of all cancers are Ras mutant (Fernández-Medarde & Santos, 2011). However, cancers with
a worse prognosis can have higher Ras-mutation rates: pancreatic cancer has a 7% survival rate
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and up to 90% mutant Ras prevalence, lung cancer has a 17.4% survival rate for all cancers and
up to 33% mutant Ras prevalence in adenocarcinomas, and colon cancer has a 64.2% survival
rate and up to 50% mutant Ras prevalence (National Cancer Institute, 2012) (Bos, 1989). The
sheer abundance of mutant Ras cancers makes the Ras protein a prominent target for intervention
in cancers. The Ras oncogene, if able to be targeted, presents an untapped opportunity to treat
late-stage and advanced cancers, some of the most hopeless cases. However, this is much easier
than it sounds. The Ras protein itself has hardly any effective inhibitors, and its rapid mutability
make it much harder to target.
Instead of targeting the Ras protein directly, cell biologists are choosing to target the
various pathways of the Ras protein. There are three pathways regulated by the Ras protein: the
Ral/mitogen-activated protein cascade (MAPK) cascade, the phosphoinositide 3-kinase (PI3K)-
dependent phosphoinositide second messenger pathway, and the Ral guanine nucleotide
exchange factor (RalGEF)/ Ral GTPases cascade (Cooper, et al., 2013). Previous research led to
the targeting of the MAPK and PI3K pathways since they can be targeted by drugs working
alone or in combination with other drugs, but this approach is limited and these pathways have
rapidly developed resistance to targeting, leading to researchers looking at other opportunities to
target mutant Ras cancers. (Cooper, et al., 2013) This opportunity may arise in the third pathway,
the RalGEF pathway, which has not been explored as much (Cooper, et al., 2013). The RalGEF
pathway contains of RalGEFs, enzymes that activate RalA/B proteins by exchanging guanine
diphosphate for guanine triphosphate, activating the RalA/B proteins. The RalA/B proteins then
engages the exocyst complex at the RalB-Sec5 subcomplex, which then promotes the activation
of TANK-binding kinase 1 (TBK1) (Cooper, et al., 2013). TBK1 inhibition is the specific point
at which intervention efforts at the research lab were targeted. Specifically, two inhibitors were
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used in the targeting efforts: BX795 and Compound II (CpII). BX795 is a commercially
available drug that inhibits TBK1 (Clark, et al., 2009). Compound II is a drug that was developed
at the research institution and also is a TBK1 inhibitor, but Compound II has a lower “off-target”
effect on other kinases, allowing it to target TBK1 more specifically (Ou, et al., 2011).
Both TBK1 and Compound II have been used in assays designed to check the sensitivity
of various cell lines to TBK1 inhibition. Unpublished data from assays run at the research
institution have shown various cell lines and their sensitivity to TBK1 inhibition. This assay
showed two cell lines which were on the sensitive end of the spectrum: H460 and HCC44, both
of which are K-Ras-mutant non-small cell lung cancer cell lines. Reverse phase protein assays
(RPPAs) ran at the lab showed the relative levels of various proteins present in the cells. Said
assays showed that H460 and HCC44 had lower levels of E-cadherin than cell lines on the TBK1
resistant side of the spectrum. HCC44 had lower levels of ß-catenin and increased levels of
ZEB1 protein, while H460 had moderate levels of both proteins. The levels of these proteins
function as indicators of a mesenchymal phenotype. A mesenchymal phenotype describes cells
that have lost attachment between one another and have changed their morphology from box-
shaped to spindle-like. This process is often associated with metastasis. (Davis, et al., 2014). The
epithelial to mesenchymal transition (EMT) is the change from an epithelial phenotype, in which
cells are boxy in shape and attached to one another, to a mesenchymal phenotype. The levels of
these markers raised an interesting area of research: the effects of mesenchymal status on TBK1
sensitivity in non-small cell lung cancer cells. In order to better explore this topic, the following
experiments were designed and run.
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4. Experiment Design
The investigation consisted of two different assays: a cytostatic and cytotoxic assay.
These two different assays serve two different purposes: the cytostatic assay is designed to
measure whether or not the drugs are stopping the multiplication of cells. The cytotoxic assay
measures the ability of the drugs to kill off the cells. Both assays give insight into the TBK1
sensitivity of the cells to the drugs. The experiment used the two aforementioned cell lines: H460
and HCC44, both of which are non-small cell lung cancer cell lines derived from
adenocarcinomas. Three different variants of each line were generated: the pBABE variant,
LKB1 variant, and LKB1 kinase dead (KD) variant. The pBABE variant is a normal cancer cell
line, but it has been stably transfected with the pBABE viral vector, which does not code for any
of the genes but rather makes it easier to manipulate the genes of the cells. The pBABE variant
acts as a control for the stable transfection of the genes into the cell. The next two variants are
both genetically modified variants of these cell line. The LKB1 variant contains an
overexpressing, functional version of LKB1, a kinase and gene that serves as a tumor suppressor.
Cells that contain an overexpressed version of LKB1 have a more epithelial phenotype than the
pBABE line, allowing observation of cells that represent more towards the epithelial end of the
EMT spectrum. Thus, the epithelial-characteristic line can be contrasted with the pBABE line,
which has been described as more mesenchymal. The pBABE cell lines then serve as the
baseline against the LKB1 lines to measure the effect of LKB1 insertion on TBK1 sensitivity.
The final variant used is an LKB1 kinase dead variant of the cell line. This version of the LKB1
gene is also overexpressed but has been rendered useless, just as the gene in cells in advanced
stage cancers might be, creating a cell that will mimic a more mesenchymal phenotype. The
LKB1-KD lines help verify that the overexpression of the active LKB1 protein is causing a more
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epithelial status and that it is causing the difference in sensitivity to TBK1 inhibition. Using these
three cell type variants from two cell types, 6 different cell lines will be tested. These cell lines
will be tested with two separate drugs: BX795 and Compound II, both of which have been
discussed in the background.
The cytostatic assay consisted of creating a drug-response curve, or DRC. In order to
create a drug-response curve, 96-well plate assay was used, using a 96-well plate (see figure 1).
Figure 1: The layout of a 96-well plate
There is a specific protocol followed during the drug-response curve assay. In order to
prevent contamination of the cells, a majority of the steps are performed in cell culture biosafety
cabinets, which protect a sterile environment for cell culture. First, cells that were being cultured
in incubators in 10 cm plates are taken and then are looked at through a microscope. If they were
not near 100% confluency (a term used to describe the amount of area the cells cover on the
plate), they were put back in the incubator to continue growing, but if they were near 100%
confluency, then the media is removed and the cells are washed 2 times with phosphate-buffered
saline (PBS). 2 mL of trypsin are then added and the plates are incubated for 5 to 10 minutes to
remove the cells from the plate. Then, 8 mL of RPMI media with 5% fetal bovine serum (FBS)
are added, which neutralizes the trypsin. This solution is then put into a tube. The cells are then
counted using an automated cell counter. If there weren’t enough cells to proceed with the assay,
the cells would be put into new 10 cm plates and then put back into incubators to grow. If there
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12
7
are enough cells, the cells would be diluted to create a solution that had 100,000 cells per mL for
H460 and 40,000 cells per mL for HCC44; the difference in cell concentration is due to the
different proliferation rates of the cells. 11 mL of cell solution would then be put into a plastic
media reservoir, which allows for the transfer of cells into the 96-well plates. The cells are then
transferred into the 96-well plate from the reservoir using a multichannel micro-pipette. 100 µL
of cell media solution, containing 4,000 cells for HCC44 or 10,000 cells for H460, are
transferred into each well. For the purposes of this assay, each cell line has two plates. The cells
were then allowed to grow for one day in incubators set at 37 degrees Celsius and 5 percent
carbon dioxide. The next day, the drugs were added to the cells. The drugs were prepared in
another 96-well plate. Each well was filled with 164 ml of media. Then, 6 wells, labeled as wells
B11 through G11, are filled with 82 µL of extra media. Then, DMSO, the compound in which
the drugs are dissolved, is added to wells B4 to B11 and G4 to G11. Then, the drug is added:
wells C11 and D11 receive BX795, and wells E11 and F11 receive Compound II, creating
concentrations of 33.33 µM. The volume of DMSO added to each well is the same as the volume
of drug added, in order to keep the concentration of DMSO in all wells equal. 82 µL is then
taken from each well that has additional media, mixing it with the media in the well to dilute the
drug, then adding those 82 µL to the wells next to the left, creating a serial dilution of one-third.
Thus, extra media is added from wells B11 through G11 to wells B10 though G10. Each serial
dilution results in a lower concentration of the compounds, but the concentration of DMSO
remains the same in wells in the same column. This serial dilution is carried on until column 4,
creating a range on concentrations. Extra media from column 4 is thrown away. This creates a
finished drug plate (see figure 2).
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Figure 2: The layout of the finished drug plate1
Media Media Media Media Media Media Media Media Media Media Media Media
Media Media Media DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO Media
Media Media Media .015[1] .046[1] .137[1] .412[1] 1.23[1] 3.70[1] 11.11[1] 33.3[1] Media
Media Media Media .015[1] .046[1] .137[1] .412[1] 1.23[1] 3.70[1] 11.11[1] 33.3[1] Media
Media Media Media .015[2] .046[2] .137[2] .412[2] 1.23[2] 3.70[2] 11.11[2] 33.3[2] Media
Media Media Media .015[2] .046[2] .137[2] .412[2] 1.23[2] 3.70[2] 11.11[2] 33.3[2] Media
Media Media Media DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO Media
Media Media Media Media Media Media Media Media Media Media Media Media
Then, 50 µL of the media from the drug plate is added to corresponding well on the cell
plate. The cell plates are then kept back in the incubator for three days, after which they are
removed and are mixed with Cell Titer Glo, which is used to measure ATP production and gives
off light in proportion to how much ATP is present. The plate is then read by a plate reader,
determining the luminescence of each well. The luminescence values are used to determine the
relative amount of living cells in each well, demonstrating how well the drug is inhibiting
cellular proliferation. Using the absorbance values, which are standardized using methods
discussed in the Results section of this paper, a drug response curve (DRC) is generated,
showing the sensitivity of the cells to the drugs.
The cytotoxic assay consisted of a qualitative assay, a 6-well plate assay. A majority of
the steps are performed in cell culture biosafety cabinets Again, the same procedure as listed
above was used to separate the cells from the 10 cm plates. Once the cell are put into the tube,
they are counted. If there are not enough cells, then they are replated and put into the incubator to
grow again. If there are enough cells, the cells are diluted to the needed concentrations at a total
volume of 26 mL. Each well in the 6-well plate is filled with 2 mL media (the concentrations of
cells necessary are 20,000 cells per well of HCC44 or 40,000 cells per well of H460) and two
1 [1] represent µM BX795, and [2] represents µM Compound II
9
plates are made for each cell line variant. The cells are allowed to grow until they are 100%
confluent. The media is then taken off from each plate and replaced with new media. This new
media contains one of 3 compounds: DMSO, BX795, or Compound II. The H460 lines have two
wells with 0.04% DMSO, one well with 2 µM BX795, one well with 4 µM BX795, one well
with 2 µM Compound II, and one well with 4 µM Compound II. The HCC44 lines have two
wells with 0.02% DMSO, one well with 1 µM BX795, one well with 2 µM BX795, one well
with 1 µM Compound II, and one well with 2 µM Compound II. The differences in
concentrations used results from the different IC50 values determined from the drug response
curve: HCC44 had lower IC50, so it required lesser concentrations than H460. The cells are then
put back into the incubator and are checked after every three days. If the plates have very little
cell death, change the media and add fresh media containing the appropriate amount of DMSO or
drugs. At the end point, after at least 2 rounds of treatment, when enough death has occurred, one
aspirates off the media and washes the cells twice with PBS. Then, one uses ice-cold menthol to
fix the cells to the plate for 10 minutes. After that, drain the cells and add 0.05% crystal violet to
stain the cells for 30 minutes. Then, properly dispose of the crystal violet and wash each well 6
times with water to wash off excess crystal violet. Each plate had a picture taken of it, and the
blue color of each well was compared by eye to those of other wells to determine the amount of
cell death present in each cell. Darker shades of blue indicate that there are more live cells and
thus less cell death, whereas lighter shades of blue indicate that there are less live cells and thus
more cell death.
5. Results
The results from the experiment are listed below. The cytostatic assay results are
discussed first, and then the cytotoxic assay. The cytostatic assay was normalized for various
10
different values. First, the values for wells treated with the drugs were normalized to average
values for the cells in DMSO: the absorbance values for DMSO wells (B4-B11 & G4-G11) were
averaged for each row individually and each of the bolded well’s absorbance values was divided
by this average value. For example, the values for wells C3 through F3 are divided by the
average value of B3 and G3 and so on. After these DMSO-normalized values were attained, the
values were again normalized to the cells growing in normal media: for example, the normalized
values for cells in wells C4-C11 are each divided by the value for the normalized value for C3,
and so on for rows D through F. The data is then plotted on a graph: the X-axis uses a log scale
to represent the concentration of the drug in µM, and the Y-axis represents the values obtained
from double normalization. The normalization process helps act as a control on the whole
experiment, taking out the effect that DMSO has on the cells themselves and leaving the effect of
the drugs on the cells. In addition, the normalization process provides data that is expressed in
simple numbers, less than or equal to 1.1, that can be compared to determine the effect of the
drugs on the cells. Thus, the normalized values show what the effects on the drugs themselves
were on the cells, taking out the effect of DMSO on the cells. In total, 12 tables of data were
attained (see figure 3 for which rows are used for absorbance values).
Figure 3: Layout of a 96-well plate. Cells in bold are used to generate curve
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12
H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12
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The tables of data listed include the double normalized data and the graphs of the data,
which include IC50 data, the concentration of drug needed to inhibit a cellular process in 50
percent of cells. The values for H460 are first, and the values for HCC44 are second.
Concentration (µM) 0 0.0152 0.0457 0.1372 0.4115 1.2346 3.7037 11.111 33.333
H460
pBABE
1
BX795 neg cont norm 1 0.98356 1.02172 1.03830 1.05783 0.50617 0.36360 0.14879 0.01204
BX795 neg cont norm 1 1.00798 1.03116 0.99805 1.03706 0.49582 0.35152 0.09567 0.00853
Cmpd II neg cont norm 1 1.01276 1.06584 1.08121 1.10837 0.93586 0.56510 0.40067 0.00440
Cmpd II neg cont norm 1 0.98941 1.01330 1.03838 1.08873 0.94114 0.58188 0.43482 0.30492
H460
pBABE
2
BX795 neg cont norm 1 0.97120 0.95902 0.96257 1.02974 0.55591 0.34979 0.13218 0.01084
BX795 neg cont norm 1 0.95161 0.96986 0.98848 1.03755 0.50707 0.37192 0.11489 0.00740
Cmpd II neg cont norm 1 0.98626 1.04627 1.03479 1.00987 0.92310 0.55729 0.34582 0.00315
Cmpd II neg cont norm 1 1.00032 1.05524 1.02239 0.97803 0.94301 0.55640 0.40191 0.00702
H460
LKB1
1
BX795 neg cont norm 1 1.01422 1.02927 1.00693 0.99611 0.52344 0.40430 0.20741 0.02633
BX795 neg cont norm 1 1.03661 1.00059 1.04080 1.00590 0.50739 0.40683 0.14840 0.02312
Cmpd II neg cont norm 1 1.04735 1.01744 1.03899 1.02468 0.92412 0.56494 0.40440 0.00493
Cmpd II neg cont norm 1 1.00131 0.99793 0.99873 1.01968 0.96352 0.62910 0.42610 0.31770
H460
LKB1
2
BX795 neg cont norm 1 1.01769 1.01595 0.96492 1.01540 0.57621 0.39369 0.18274 0.02690
BX795 neg cont norm 1 1.01668 1.04073 1.00959 1.03882 0.62567 0.41842 0.15120 0.02303
Cmpd II neg cont norm 1 0.99946 1.00489 1.05080 1.07983 1.01206 0.63254 0.41769 0.00393
Cmpd II neg cont norm 1 1.02365 1.01336 1.00887 1.05333 0.97664 0.64197 0.43532 0.00793
H460
LKB1-
KD 1
BX795 neg cont norm 1 0.93621 1.01006 1.01286 0.97351 0.36270 0.28845 0.12150 0.01349
BX795 neg cont norm 1 0.96843 1.00819 0.90525 0.95963 0.37362 0.28003 0.09391 0.01053
Cmpd II neg cont norm 1 0.96833 1.02872 1.05740 1.00134 0.66236 0.36501 0.27831 0.00306
Cmpd II neg cont norm 1 0.94188 0.99273 1.01092 0.96580 0.82319 0.37990 0.30403 0.00617
H460
LKB1-
KD 2
BX795 neg cont norm 1 0.98647 0.99820 1.04713 1.01077 0.39464 0.30070 0.15149 0.01505
BX795 neg cont norm 1 0.99364 1.00755 1.01656 0.95136 0.36995 0.28234 0.11008 0.00932
Cmpd II neg cont norm 1 0.97695 1.00214 0.95595 0.94231 0.67752 0.38118 0.28432 0.00259
Cmpd II neg cont norm 1 0.97143 0.99860 0.94285 0.98338 0.66643 0.37971 0.29916 0.00650
H460 DRC - BX795 H460 DRC – Compound II
-2 -1 0 1 2 0.0
0.5
1.0
Log10 Drug [ ] µM
H460 + pBABE + BX795 H460 + LKB1 + BX795 H460 + LKB1-KD + BX795
BX795 IC50 (+ pBABE) = 1.70 µM
BX795 IC50 (+ LKB1) = 1.98 µM
BX795 IC50 (+ LKB1-KD) = 0.95 µM
-2 -1 0 1 2 0.0
0.5
1.0 H460 + pBABE + Compound II H460 + LKB1 + Compound II
H460 + LKB1-KD + Compound II
Cmpd II IC50 (+ pbabe) = 5.88 µM Cmpd II IC50 (+ LKB1) = 7.73 µM Cmpd II IC50 (+ LKB1-KD) = 2.94 µM
Log10 Drug [ ] µM
12
Concentration (µM) 0 0.01524 0.04573 0.13717 0.41152 1.23456 3.70370 11.1111 33.3333
HCC44
pBABE
1
BX795 neg cont norm 1 0.95182 0.91345 0.87610 0.64323 0.14863 0.02815 0.00780 0.02512
BX795 neg cont norm 1 0.99263 0.96508 0.89113 0.59972 0.16480 0.01608 0.00389 0.02076
Cmpd II neg cont norm 1 0.94151 0.93747 0.85827 0.54909 0.22645 0.09981 0.00240 0.00124
Cmpd II neg cont norm 1 0.96806 0.95356 0.81018 0.58999 0.26824 0.10527 0.00956 0.00485
HCC44
pBABE
2
BX795 neg cont norm 1 0.88060 0.84192 0.83030 0.58139 0.15285 0.02600 0.00962 0.02681
BX795 neg cont norm 1 0.97348 0.94232 0.90512 0.66977 0.16682 0.03164 0.00478 0.02817
Cmpd II neg cont norm 1 0.95433 0.94738 0.90415 0.60552 0.25320 0.08821 0.00867 0.00149
Cmpd II neg cont norm 1 0.93927 0.94274 0.92799 0.66471 0.34715 0.13368 0.04043 0.00562
HCC44
LKB1
1
BX795 neg cont norm 1 0.93656 0.88222 0.85490 0.74851 0.52492 0.24442 0.01584 0.11927
BX795 neg cont norm 1 0.94189 0.91926 0.85040 0.74517 0.51320 0.22661 0.01046 0.11903
Cmpd II neg cont norm 1 0.89163 0.88262 0.80438 0.62695 0.38002 0.26105 0.06274 0.00274
Cmpd II neg cont norm 1 0.87759 0.85433 0.81648 0.64881 0.41146 0.27231 0.10540 0.00726
HCC44
LKB1
2
BX795 neg cont norm 1 0.83700 0.84675 0.84353 0.69053 0.51115 0.17409 0.01533 0.11717
BX795 neg cont norm 1 0.89465 0.87673 0.82222 0.70354 0.54096 0.30614 0.01508 0.11078
Cmpd II neg cont norm 1 0.90148 0.90079 0.86677 0.64049 0.41924 0.28035 0.06898 0.00263
Cmpd II neg cont norm 1 0.89007 0.87717 0.85540 0.72541 0.46877 0.31813 0.15985 0.00683
HCC44
LKB1-
KD 1
BX795 neg cont norm 1 0.92521 0.88598 0.83302 0.63865 0.14958 0.03460 0.00882 0.01505
BX795 neg cont norm 1 0.97602 0.84927 0.78791 0.60570 0.1153 0.02806 0.00392 0.01115
Cmpd II neg cont norm 1 1.00832 0.92705 0.80919 0.52030 0.23376 0.09320 0.00224 0.00110
Cmpd II neg cont norm 1 0.98802 0.90272 0.79397 0.52815 0.24441 0.10108 0.00729 0.00523
HCC44
LKB1-
KD 2
BX795 neg cont norm 1 0.78187 0.84149 0.76611 0.64879 0.14569 0.02464 0.010604 0.01580
BX795 neg cont norm 1 0.85985 0.93369 0.81514 0.69233 0.15743 0.03568 0.003437 0.01669
Cmpd II neg cont norm 1 0.99238 0.98010 0.82994 0.54603 0.21700 0.09719 0.002761 0.00147
Cmpd II neg cont norm 1 0.97113 0.92593 0.76923 0.59189 0.30591 0.10262 0.007124 0.00529
HCC44 DRC – BX795 HCC44 DRC – Compound II
The cytotoxic assay does not provide a number output, but rather one can tell the relative
amount of dead cells through image analysis. By looking at the shade of the stain, one can tell
qualitatively how much the cells died and determine the effectiveness of the drug, as darker
shade indicates less cell death and a lighter shade indicates greater cell death. The pictures that
-2 -1 0 1 2 0.0
0.5
1.0
Log10 Drug [ ] µM
HCC44 + pBABE + BX795 HCC44 + LKB1 + BX795 HCC44 + LKB1-KD + BX795
BX795 IC50 (+ pBABE) = 0.56 µM BX795 IC50 (+ LKB1) = 1.40 µM BX795 IC50 (+ LKB1-KD) = 0.62 µM
-2 -1 0 1 2 0.0
0.5
1.0
Log10 Drug [ ] µM
HCC44 + pBABE + Compound II HCC44 + LKB1 + Compound II HCC44 + LKB1-KD + Compound II
Cmpd II IC50 (+ pBABE) = 0.61
µM Cmpd II IC50 (+ LKB1) = 1.35 µM Cmpd II IC50 (+ LKB1-KD) = 0.48 µM
13
were taken of the plates are listed, along with a caption describing the variant of the cell line and
when it was stained. The pictures are below, with H460 first and then HCC44. The layout of the
plates is shown in the tables below (first for H460 and then for HCC44):
Figure 4: The layouts for the plates (H460 and HCC44)
0.04% DMSO 4 µM BX795 4 µM Compound II
0.04% DMSO 2 µM BX795 2 µM Compound II
0.02% DMSO 2 µM BX795 2 µM Compound II
0.02% DMSO 1 µM BX795 1 µM Compound II
6. Analysis
The H460 DRC had interesting results. The IC50 values of the pBABE lines for BX795
(1.70 µM) was not much different than that of the LKB1 variant (1.98 µM), and the curves for
both lines seem to indicate that they have similar sensitivity to BX795. The LKB1-KD line,
however, has a much lower IC50 value (0.95 µM), almost half of the pBABE and LKB1
variants. Comparing the more epithelial variant (LKB1) and the more mesenchymal variants
(LKB1-KD and pBABE), one can see that the variants displaying a more mesenchymal
phenotype are more sensitive to TBK1 inhibition through BX795 than the variant with a more
H460 Set 1
pBABE
H460 Set 1
LKB1
H460 Set 1
LKB1-KD
H460 Set 2
pBABE
H460 Set 2
LKB1
H460 Set 2
LKB1-KD
HCC44 Set
1 pBABE
HCC44 Set
1 LKB1
HCC44 Set 1
LKB1-KD
HCC44 Set 2
pBABE
HCC44 Set
2 LKB1
HCC44 Set
2 LKB1-KD
14
epithelial phenotype is, though the pBABE variant is not that much more sensitive to TBK1
inhibition than the LKB1 variant. This could be because the overexpression of LKB1 (as in the
LKB1 lines) does not affect the levels of LKB1 that much from the base state and thus does not
affect the sensitivity of the cell to TBK1 as much, but the overexpression of an inactive form of
LKB1 (as in the LKB1-KD lines) causes the cells to become much more sensitive to TBK1
inhibition.
The results from the Compound II DRC for H460 also show similar results: there is not
that much difference in between the IC50 values for the pBABE (5.88 µM) and LKB1 (7.73
µM). However, the LKB1-KD line has an IC50 (2.94 µM), more than half that of the LKB1 line
(7.73 µM). So, just as the values for the H460 BX795 DRC shows, the LKB1 overexpression
does not have that much of an effect on TBK1 sensitivity, though the values for the LKB1-KD
line are much lower, indicating that the overexpression of the inactive form has a much greater
effect on the phenotype than the overexpression of the active form. The data from both DRCs
supports the hypothesis, since the values for the more mesenchymal lines (LKB1-KD and
pBABE) are less that the values for the more epithelial lines (LKB1), even though the pBABE
values are not much lower than those of LKB1.
The HCC44 lines show a much greater sensitivity than do the H460 lines, and a different
pattern exists in the IC50 values. The IC50 for BX795 of the pBABE line (0.56 µM) was
extremely close to that of the LKB1-KD line (0.62 µM). However, there is a large disparity
between the BX795 IC50 the pBABE line (.56 µM) and the LKB1 line (1.40 µM). The IC50 of
the LKB1 variant is much higher than that of the pBABE or LKB1-KD variants. Thus, the data
supports the hypothesis, since the cells overexpressing LBK1, which have a more epithelial
phenotype, are less sensitive to TBK1 inhibition. The Compound II DRC data matches up with
15
that of the BX795 DRC: both the pBABE line (0.62 µM) and LKB1-KD line (0.48 µM) have
similar IC50s, whereas the LKB1 line has a much higher IC50 (1.35 µM). The data show that the
more epithelial variant is much less sensitive to TBK1 inhibition than the more mesenchymal
variants. In addition, LKB1 overexpression has a much greater effect in the HCC44 cells, which
have a much higher IC50 value for the LKB1 lines, whereas the overexpression of an inactive
form of LKB1 does not have much of an effect on TBK1 sensitivity, as seen through the similar
IC50 values of the pBABE and LKB1-KD lines.
The cytotoxic binary assay also shows the effects of BX795 and Compound II on the
cells by showing how much they are killed rather than how much they stop growing. The H460
lines presented a challenge in that they did not show much death upon one week of drug
treatment (as shown by Set 1). There is some cell death present in the cells, but only discernible
in the 4 µM BX795 wells, and only by a little bit. A second week of treatment with fresh media
produced greater results, allowing for images which can be better distinguished, though only
slightly so. Though it is still hard to tell, the Set 2 plates show greater cell death in the pBABE
and LKB1-KD plates versus those in the LKB1 plates in the wells treated with 4 µM BX795,
since the pBABE and LKB1-KD plates are lighter, indicating greater cell death. The other wells
are still too close in color to be able to make a distinction regarding the effects of the drugs on
the cell lines. This cell line might be more resilient to the drug treatment, and the cytotoxic
effects of the drug may be limited. However, the greater resistance to treatment suggested by the
BX795 DRC and Compound II DRC of the cell line may account for this phenomenon, thus
meaning that the cell line may require more treatment with a higher concentration of drugs
before showing much cell death.
16
The HCC44 cytotoxic assay results mirrored those from the DRC: the pBABE and
LKB1-KD lines are much lighter in color than the LKB1 cell line in the wells treated with
BX795 and 2 µM Compound II, again showing that the mesenchymal type cells are more
sensitive to TBK1 inhibition than are the epithelial type cells. At the same time, the HCC44 lines
show a much greater difference in color from being treated with the drugs than did the H460
lines, a result of HCC44’s lower IC50 when compared to H460, thus showing that the line is
more sensitive to TBK1 inhibition. Still, both plates do have wells that were not able to be
distinguished very well from one another. The data from the H460 cytotoxicity assay is
inconclusive: the wells are so close to each other in color that they cannot be distinguished.
However, the H460 assay does not contradict the hypothesis, and the HCC44 assay supports the
hypothesis.
7. Discussion, Extension, and Conclusion
The results from the experiments support the hypothesis, that cells with a more
mesenchymal phenotype are more sensitive to TBK1 inhibition than are cells with a more
epithelial phenotype. The data from the DRC show that the LKB1-KD and pBABE lines, which
are more mesenchymal in nature, show greater sensitivity to BX795 and Compound II than the
LKB1 line, which is more epithelial in nature. Also, the cytotoxic assays tends support the
hypothesis, as the cytotoxic assays show a lighter shade in the LKB1-KD and pBABE lines than
the LKB1 lines, which are a much darker shade of blue, even though the results from the H460
assay are inconclusive.
One of the improvements that could be made to this investigation is performing another
trial of the cytotoxic assay. The H460 lines could be treated with higher concentrations of the
drug to see a greater effect of the drug on the H460 lines. This would create a lighter shade of
blue that would be much easier to read and make it easier to see the differences between the
17
various drug concentrations and different cell line variants. The same improvement could be
made to the HCC44 lines as well, as some of the wells are very similar in color across all plates,
such as the 1 µM Compound II wells. This would help provide more conclusive results for the
experiment. These are the improvements that could be made to the current experimental design.
However, further studies and experiments are needed to determine whether or not TBK1
inhibition is a viable method for treating more mesenchymal-type cancers. Further experiments
would also be required to determine the efficacy of TBK1 inhibition at killing Ras mutant cancer
cells. One further extension of the cytotoxic assay would be to quantify the results obtained from
the experiment. This would involve repeating the cytotoxic assay, but once the plates are stained,
the stains would be dissolved in acid and quantified, providing numerical values that could be
used to compare the cell death present in the wells. This method would make it easier to check
the amount of cell death present on the plates, providing more certainty to the results obtained
from this assay. Another set of assays that could be used to determine the mesenchymal status of
the cells are Western Blots. A Western Blot involves taking protein lysate from the cells,
separating the protein lysate from the cells based on size of protein fragments in a
polyacrylamide gel, then transferring the protein onto a membrane and blotting it with primary
antibodies to detect the presence of proteins on the membrane. The membrane is then treated
with secondary antibodies, which are tagged with a chemiluminescent indicator. The membrane
is then put next to photography film in a dark room, and this film is developed to present a
readout of the various protein levels. This assay could be used to detect the relative levels of
various proteins within the cells. There are two different Western blots that could be run: one
would involve using the cell line variants without any drug treatment and measuring the various
levels of mesenchymal-state and epithelial-state markers, such as E-cadherin, vimentin, ß-
18
catenin, and N-cadherin. This would help establish whether the cell line variants are more
epithelial or more mesenchymal, allowing for a better interpretation of the data from this
analysis. Another Western Blot assay would involve the same set-up as the cytotoxic assay, but
instead of dying the cells, the cells would be lysed and protein lysate would be collected. The
protein lysate could be used to determine the effects that TBK1 has on a cellular level, showing
which pathways are being inhibited and how this relates to the mesenchymal or epithelial state of
the cell. Currently, the data suggests a correlation between the mesenchymal status of a cell and
its sensitivity to TBK1 inhibitors, but there is not an understanding of what makes the cells more
sensitive to TBK1 inhibition, so such an assay would illuminate what is occurring at the cellular
level. Finally, this experimental analysis only looked at two cell lines that had been selected
based on their characteristics. Expanding the research to include variants of other cell lines
would provide even greater data into this phenomenon, providing greater insight. In order to test
the real world efficacy of TBK1 inhibition and its correlation to mesenchymal status, mouse
studies would need to be conducted. Mice would need to be implanted with tumors of different
mesenchymal and epithelial characteristics and treated with TBK1 inhibitors such as BX795 and
Compound II, in order to see the correlation of the data in an animal setting
Though these are all further extensions of the experiment, the data from this assay
suggests a new method of treating cancer. The cell lines studied in the assay were all K-Ras
mutant cancer cell lines, and this assay seems to show that TBK1 inhibition may work in
combating K-Ras mutant cancers, which have low survival rates, as mentioned in the
introduction. At the same time, mesenchymal type cells are associated with late-stage and
advanced cancers, which have undergone metastasis. TBK1 inhibition, based off the results of
this experiments, may hold a promise in treating such types of cancers. The possibilities for this
19
method of treatment are bright: more experiments need to be done to ascertain the efficacy of
TBK1 inhibition for treating mesenchymal-type cancers.
In conclusion, this investigation has provided evidence supporting the hypothesis, that
cells with more mesenchymal phenotype are more sensitive to TBK1 inhibition than are cells
with a more epithelial phenotype. This research suggests that there could be applications to treat
advanced and late-stage cancers, though further research is required to test the applications of the
drug in this setting. The results of this experiment may lead to new discoveries to combat mutant
Ras cancers, helping to provide hope to those who have very little.
20
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