Drug Synergy Study of Chemo-resistant Prostate

Cancer Cells

Tausif Khan

Dr. Arif Hussain

Toni Ireland

Intern Mentor I

May 5, 2019

Abstract:
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Prostate cancer is the second most important cancer for men in the US. In this study of

prostate cancer, we asked whether the combination of drugs MK2206 and Lapatinib are

synergistic to treat LNCaP, C4-2, and C4-2/DocR, a docetaxel resistant cell line. We adopted the

Chou-Talalay method to find whether or not these drugs are synergistic in these cell lines. We

found that the combination of MK2206 and Lapatinib has great potential for being synergistic for

these cell lines. If validated by confirmatory experiments, combination of these two drugs will

have a great therapeutic promise to treat prostate cancer.

Introduction:
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Approximately 1 out of 9 people are diagnosed with prostate cancer and is the second

most important cancer in men in the United States. People over 60 are most affected by this

malignancy; however, people can get prostate cancer even earlier in their life. People who get

prostate cancer are initially treated with hormone therapy, but eventually, hormone therapy fails

when prostate cancer cells become hormone refractory or resistant to hormonal treatment. The

patient then receives chemotherapy; however, prostate cancer cells eventually become resistant

to the chemotherapy and they will be categorized as chemo-resistant. Targeted small molecule

therapy is one of the favorite options for treating prostate cancer. This therapy blocks one or

more specific pathways and leads the cancer cells to death; yet, it is not uncommon that cancer

cells find alternate pathways to survive and grow. Targeted therapy often depends on the cellular

response against a particular drug which needs a clear understanding of the cell’s biological

pathways to survive and grow. Scientists discovered that sometimes targeting cells with two or

more small molecule inhibitors show better results with the tolerable level of cell drug toxicity.

MK2206, a type of drug that is a small molecule inhibitor, is proven effective in successfully

inhibiting AKT phosphorylation, or the activation of the protein AKT, and blocking the PI3K-

AKT pathway. Lapatinib, another small molecule inhibitor, works by blocking two receptors on

a prostate cancer cell called erbB1 and erbB2. The reason for targeting these two receptors is to

block the EGFR activated downstream pathways which are the main pathways a prostate cancer

cell can use to get activated. In this study, LNCaP (control prostate cancer cell line), C4-2

(control prostate cancer cell line), and C4-2/DocR (Docetaxel Resistant C4-2 cell line or the

chemo-resistant cell line) cells were treated with MK2206 and lapatinib both as a single agent

and in combination to test whether these two drugs are synergistic on these cell lines. The

purpose of this study is to find whether the combinations of two drugs MK2206 and lapatinib are
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synergistic in treating prostate cancer cells. This paper will clarify the importance of knowing the

molecular genetics of prostate cancer, why use MK2206 and Lapatinib, why they have the

potential to be synergistic, and what the Chou-Talalay method is and its application.

Review of Literature:

To conduct a synergy study in prostate cancer cells, it is important to understand the

genetics of the cancer cells. The genes that are important in prostate cancer are RNASEL,

MSR1(macrophage scavenger receptor 1), AR(androgen receptor), CYP17(cytochrome P-450)

and STD5A2 (Nelson W. G et. al. 2003). Knowing the genetics of a cell with prostate cancer can

show which drugs would be most effective in suppressing any pathways that it would take to

survive. Hormone dependent prostate cancer cells survive on a major pathway called the

androgen receptor pathway or AR pathway. Prostate cancer cell line pathways suggests that

androgen receptor is the main protein that activates many other proteins that leads to prostate

cancer. Most prostate cancer cell lines are androgen dependent, but there are cases where the

cells can be androgen independent. In some cases, prostate cancer cells lose a tumor suppressor

gene called PTEN that results in increased AKT activation and carry on the cell signaling. To

realize if cells are cancerous or not, there are a few distinctive characteristics or hallmarks. Self-

sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of

programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and

tissue invasion are the 6 identifiable hallmarks (Cree, I. A et. al. 2016). A number of genes is

responsible to produce signals for cells death after its job is done. However, sometimes apoptosis

(programmed cell death) signaling pathways are disrupted for various reasons, so then the cells

survive and are considered cancerous due to the evasion of apoptosis. Another important

pathway the cells can take is the ErbB signaling pathway. ErbB receptor family members are
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basically the receptor tyrosine kinase located on the plasma membrane of the cell. The mutation

and/or amplification of these receptors can make them constitutively active, thus producing a

cascade of signaling throughout the cells and signals for promoting cell growth.

Lapatinib and MK2206 are two small molecular inhibitors that are cytotoxic for cancer

cells. In other studies, the effects of MK2206 were tested on lung, breast, and ovarian cancer.

Different cell lines were treated with MK2206 in combination with different drugs like erlotinib

or lapatinib. In several cases, it is found that the combination of drugs has synergistic effects not

only in wild type cells but also in chemoresistant cells (http:// mct.aacrjournals.org

/content/9/7/1956.long). Different drugs that minimize the activation of receptor signaling

pathways have already been tested by other scientists and researchers. A number of antibodies

(trastuzumab, cetuximab) and tyrosine kinase inhibitors (lapatinib, erlotinib, gefitinib) are FDA

approved and commercially available that can target these ErbB receptors (Hynes, N. E et. al.

2009). Researchers are working with lapatinib with targets ErbB1 and ErbB2 receptors to

inactivate them. However, since the PI3 kinase and AKT pathway gets hyperactivated, it is

crucial to block both pathways simultaneously which researchers are utilizing with a

combination of MK2206 and lapatinib. MK2206 is being used because it targets a pathway

called the PI3K-AKT pathway, but first, the mechanisms of the PI3K-AKT pathway must be

defined in order to understand why it is the main target. PI3K stands for phosphatidylinositol - 3-

kinases, a family of enzymes that are involved with many cellular functions especially cell

growth, proliferation, differentiation and other biological activities (Georgescu, M. M et. al.

2010). This is important to understand because this is one on the pathways that is being targeted

by MK2206. PTEN is an important protein that has influence over the PI3K-AKT pathway.

Expression of PTEN induces a brake or a marked decrease of cell proliferation by arresting cell
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cycles in the G1 phase of the cell cycle. Currently, there is focus on working with LNCaP and

C4-2/DocR cell lines which have PTEN mutations. It is imperative to understand the effects of

the PTEN mutation in cells so it is taken into consideration when treating cells.

When determining the synergism of two drugs in cell lines, there are some challenges of

the drug combination in cancer therapy. In vitro screening of a large number of drug

combinations also poses a huge challenge because of the high number of permutations of the

drug combinations (Azvolinsky Anna et. al. 2018). In vitro drug, combination screening is

expensive, labor extensive, and high costs are involved in it. Therefore, small scale combination

screenings are done by academic labs or pharmaceutical companies (Azvolinsky Anna et. al.

2018). A phase 1 study is being conducted to treat HER2 positive breast cancer with a

combination of MK2206 and lapatinib to find out the maximum tolerated dose (MTD) for both

the drugs. MK2206 and lapatinib show potential in treating docetaxel resistant prostate cancer.

Lapatinib is is a “small molecule inhibitor” which targets the EGFR pathway. Unlike other

drugs, lapatinib can “enter into the cells and bind to the intracellular domain of the tyrosine

kinase receptor” and completely blocks autophosphorylation reaction and halts the downstream

pathway of EGFR mediated signaling which is why it’s a dual tyrosine kinase inhibitor (Paul B.

et. al. 2008). Lapatinib can be taken orally and combined with other cancer drugs, especially

capecitabine, which performs better than single agents for the treatment of metastatic drug-

resistant breast cancer. The mechanisms of targeted cancer therapy are by using drugs or other

substances to block the growth and spread of cancer by interfering with specific molecules

("molecular targets") that are involved in the growth, progression, and spread of cancer (Targeted

Cancer Therapies). They differ from chemotherapy in a way that they act on a specific molecule

in the cancer cells whereas chemotherapy targets all dividing cancer cells as well as normal cells.
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Ting-Chou described a methodology for a drug combination study using equations like

the Michaelis-Menten Hill, Henderson-Hasselbalch, and Scatchard equations (Drug Combination

Studies and Their Synergy Quantification Using the Chou-Talalay Method). Ting-Chou

described the drug combination and based this methodology on “the median-effect equation

which is derived from the mass action law principle”. From this, he coined the term combination

index (CI) which can be additive (CI=1), synergistic (CI<1), or antagonistic (CI>1) to cells

(http://cancerres. aacrjournals.org /content/70/2/440.long). When testing the effect of two drugs

against a drug-resistant prostate cancer cell line, the success of the trial is determined by a

combination drug index. The combination drug index from the Chou-Talalay method was

considered the standard to measure how synergistic these two drugs are. The results were then

analyzed with statistical software to find the mean ± standard deviation (SD). The dose-effect

relationship or combination index (CI) was determined by using the Chou-Talalay method. MTT

assay is a suitable tool to study drug resistance of the cells. The MTT assay can be used for the

study of cross-resistance between related and unrelated drugs, measuring drug sensitivity to

predict clinical outcomes, sensitivity testing of a new drug, drug screening on cell lines, and

testing drug combinations on cell lines. This is the main assay that is being conducted in order to

find the drug combination index to see if Mk2206 and lapatinib are synergistic or not.

Combination of drugs for targeted therapy is a relatively new concept. It has been proved

that MK2206, in combination with lapatinib is proven to be more effective to control breast

cancer and lung cancer. To explore this concept in treating prostate cancer would be an

interesting finding because prostate cancer cells are heterogeneous in nature, these particular two

drugs may not be effective for all kinds of prostate cancer cell lines. Since LNCaP has a PTEN

mutation and expresses AKT constitutively, it will be prudent to use MK2206 to treat LNCaP
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cell line and its derivatives. On the other hand, research showed that blocking AKT pathway

might activate ERbB signaling pathways. Therefore, targeting both Mk2206 and lapatinib will

theoretically make sense. If the synergistic effects of these drugs are found, it will open an

avenue for further research with other cell lines as well as different kinds of cancers.

Research Methods and Data Collection:

Cell lines: androgen dependent prostate cancer LNCaP, androgen independent C4-2, and

docetaxel resistant C4-2/DocR cell lines have been used to conduct the study. C4-2 is an

androgen independent cell line from parental LNCaP. C4-2 cell line is exposed to the selective

pressure for a long time to the create docetaxel resistant (8 nM) C4-2/DocR cell line.

Cell culture medium: RPMI 1640 supplemented with 10% fetal bovine serum was used as a

complete culture medium. C4-2/DocR cells were constantly cultured in 8nM docetaxel medium

to maintain the selective pressure of the drug. 1% penicillin-streptomycin was added to the

medium to avoid any microbial contamination.

Drugs, chemicals, and reagents: AKT inhibitor MK2206 was obtained from Merck (Kenilworth,

N.J. 07033), erbB1 and erbB2 dual inhibitor lapatinib was obtained from Selleck Chemicals, and

Thiazolyl Blue Tetrazolium Bromide (MTT) was obtained from Sigma-Aldrich.

Cell growth inhibition assay: 2500-3000 cells per well were plated in 96 well plates. After being

attached to the surface, they were treated with drugs and incubated for 72 hours. Cells were then

subjected to treatment with 5 mg/ml MTT solution and were incubated for 3 hours. The

tetrazolium crystals were dissolved with isopropanol and read at 570 nm wavelength in a plate
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reader. The readouts were used either to calculate the growth inhibition at IC50 level or to

determine the fraction of cells affected by the drugs.

Chou-Talalay method: This method is derived from the median-effect equation (MEE) of the

mass-action law developed by Chou (1974). The fraction of the cells affected by two or more

drugs used to treat cells can be calculated by using his formula which gives rise an index called

combination index (CI). If the CI< 1, the drugs deemed synergistic; if CI=1, the effect is

considered additive and in CI>1, then the effect is considered as antagonistic. Calcusyn 2.1 was

used to obtain Isobologram and Fa-CI graph.

Fraction affected (Fa) calculation: To calculate the fractions affected by the combination of two

drugs, the following formula (Bijnsdorp et. al. 2011) is used FA= 1- (% growth/100), where the

percent of growth is calculated from the growth inhibition results from the experiment.

Isobologram: the diagram shows the two drugs used in a constant ratio and whether their

combination yields a synergism or antagonism.

Fa-CI plot: An effect oriented plot shows the drug combination index against the fraction

affected at different levels.

Statistical Analysis: The mean, standard deviation, standard error of mean, and fraction affected

values were calculated by using software GraphPad Prism 7.02 or MS excel.

Results and Data Analysis:

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In order to establish the comparative resistance of C4-2/DocR cells compared to LNCaP and C4-

2 cells the growth inhibition assay or MTT assay was performed. As demonstrated in Fig 1, C4-

2/DocR is about 50 times more resistant to docetaxel than LNCaP and more than 80 times

resistant than C4-2 cell line. Table 1 shows IC50 values of all the cell lines involved in this study

against two drugs MK2206 and lapatinib. It is clear that lapatinib is three times of strength than

MK2206 to get the cells population down to 50 percent. Table 2 is an overall summary of the

combination index that indicates the synergism level of the combination of two drugs and

whether they are synergistic, additive or antagonistic. The fraction affected of the cells

considered 0.5, 0.75, 0.9 which indicates 50, 75 and 90 percent of the cells killed by the drug

combinations. It is noted that drug combinations were mostly synergistic for all the cell lines in

this study. A slight antagonism in 90 percent cell death in LNCaP cells show that, at times when

drug doses are used in a higher concentration, they might lose their stoichiometry, and hence

show an antagonistic behavior towards each other. Since MK2206 and lapatinib have been used

in a constant ratio (1:3), an Isobologram has been deduced to show if the combination index is

synergistic around the doses of the drugs in combination study. Isobologram generated from the

experiment show a synergism of the drug combinations for all the cell lines (Fig. 2A, 2C and

2E). Fa-CI plots (Fig. 2B, 2D, and 2F) show that the drug synergy is maintained throughout the

fraction affected (Fa) at all levels. It suggests that the combination of MK2206 and lapatinib are

not only effective for androgen dependent LNCaP but it also can be used for androgen

independent C4-2 or even a chemo resistant C4-2 cell line.

Discussion and Conclusion:

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Chemo-resistant prostate cancer cell line is a massive challenge for physicians and are

often treated with small molecule inhibitors like MK2206 and Lapatinib. Our study shows a

major potential to explore the combination of these two drugs clinically. Since LNCaP and its

derivatives C4-2 and C4-2/DocR have mutated AKT, it was expected that MK2206 would

effectively block the pathway and reduce its growth. When the AKT pathway is blocked, then

the ErbB mediated pathway gets activated which can be blocked by Lapatinib. However, it

wasn’t known whether the combination of MK2206 and lapatinib would effectively show

synergism in these cell lines. Our research, showed this to be evident that MK2206 and lapatinib

together work synergistically in these prostate cancer cell lines, especially in the chemoresistant

cell line. It is worthy to mention that stoichiometry plays an important role to successfully

conduct these synergy experiments. In another experiment (data not shown), results show that a

much higher or lower concentration would disrupt the actual stoichiometry and therefore using

very high or low concentration of drugs may not be as effective in developing synergism. The in

vitro synergism study needs to be validated by performing further experiments like apoptosis

assay and cell cycle distribution assay. Before the results can be used in clinical trial, an in vivo

experiment needs to be performed to find both the efficacy of the combinations and the toxicity

of the two drugs used together.

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Appendices:

Table1: IC50 Values for different cell lines for MK2206 and

Lapatinib

Cell lines IC50 (uM)

MK2206 Lapatinib

LNCaP 0.7 ± 0.07 6±0.2

C4-2 2.55 ± 0.106 5.6±0.3

C4-2/DocR 1.95 ± 0.106 6.15±0.05

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Cell lines and

Fractions affected Combination Index (CI) Synergy Level Desciption

(fa)

LNCaP

0.5 0.465 ± 0.017 +++ Synergism

0.75 0.499±0.119 +++ Synergism

0.9 1.153±0.523 - Slight antagonism

C4-2

0.5 0.45±0.197 +++ Synergism

0.75 0.17±0.033 ++++ Strong synergism

0.9 0.23±0.045 ++++ Strong synergism

C4-2/DocR

0.5 0.48±0.129 +++ Synergism

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0.75 0.322±0.026 +++ Synergism

0.9 0.354±0.051 +++ Synergism

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