Conflicting evidence for the role of JNK as a target in breast cancer cell proliferation: Comparisons between pharmacological inhibition and selective shRNA knockdown approaches

Abstract As a target, the JNK pathway has been implicated in roles including cell death, proliferation, and inflammation in variety of contexts which span cardiovascular disease, neurodegenerative pathologies, and cancer. JNK1 and JNK2 have recently been demonstrated to function independently, highlighting a new parameter in the study of the JNK pathway. In order for JNK1 and JNK2‐specific roles to be defined, better tools need to be employed. Previous studies have relied upon the broad spectrum JNK inhibitor, SP600125, to characterize the role of JNK signaling in a number of cell lines, including the breast cancer cell line MCF‐7. In line with previous literature, our study has demonstrated that SP600125 treatment inhibited c‐Jun and JNK phosphorylation and MCF‐7 proliferation. However, in addition to targeting JNK1, JNK2, and JNK3, SP600125 has been previously demonstrated to suppress the activity of a number of other serine/threonine kinases, making SP600125 an inadequate tool for JNK isoform‐specific roles to be determined. In this study, lentiviral shRNA was employed to selectively knockdown JNK1, JNK2, and JNK1/2 in MCF‐7 cells. Using this approach, JNK phosphorylation was fully inhibited following stable knockdown of respective JNK isoforms. Interestingly, despite suppression of JNK phosphorylation, MCF‐7 cell proliferation, cell cycle progression, or cell death remained unaffected. These findings raise the question of whether JNK phosphorylation really is pivotal in MCF‐7 cell growth and death or if suppression of these events is a result of one of the many off‐targets cited for SP600125.

similarity across family members that make specificity difficult to achieve without off targets and side effects. 4,5 The JNK family consists of three proteins, JNK1, JNK2, and JNK3, with JNK1 and JNK2 expressed ubiquitously while JNK3 expression is restricted to the brain, heart, and testis. 6 JNK1 and JNK2 has recently been demonstrated to function independently therefore before success can be made in developing more selective drugs that target this pathway, there has to be a clearer understanding of the JNK1-and JNK2-specific roles. While inhibitors exist that suppress the JNK pathway, so far they display poor potency 7 and are nonselective. 8 Many studies investigating JNK as a target have used pharmacological inhibition of the signaling pathway by SP600125. In addition to targeting JNK1, JNK2, and JNK3, SP600125 has been previously demonstrated to suppress the activity of a number of other serine/threonine kinases, 9 which makes SP600125 an inadequate tool for JNK1-and JNK2-specific roles to be determined. Success in this area has been best illustrated in studies using JNK1 and JNK2 knock out mouse models. Studies have also been shown to function differently in cell death induced by ultraviolet (UV) radiation. 11 JNK1 knockout resulted in partial protection against UV radiation in MEFs, whereas JNK2 À/À MEFs behaved similarly to wild type (WT). These findings demonstrate that JNK isoforms can function differently and independently in both cell death and cell growth processes in MEFs, however, a detailed understanding of JNK1 and JNK2 function needs to be more widely explored in human cell models. This has been best exemplified in the cancer field where different roles for JNK isoforms have been demonstrated using a variety of different tools.
In hepatocellular carcinoma (HCC), JNK has been linked to both the pathogenesis and poor prognosis of the disease. 12 In this study, lentiviral approaches were used to stably knockdown JNK1, JNK2, and JNK1/2 in the HuH-7 HCC cell line to investigate the effects of JNK knockdown on cell proliferation. JNK1 and JNK1/2 knockdown produced a decrease in proliferation, whereas JNK2 knockdown had no significant effect on proliferation which translated to the development of smaller tumors when implanted into nude mice, clearly linking JNK1 but not JNK2 to the tumorigenesis of HCC cells. Other studies have used the JNK inhibitor, SP600125, to pharmacologically inhibit JNK activity to implicate this pathway in breast cancer cell models. 13,14 Studies carried out in the MCF-7 breast cancer cell line have shown that the JNK pathway is required for cell death in response to UV 15 and taxol 16 and also for MCF-7 proliferation and cell cycle progression. 15 Given the selectivity issues of SP600125, the roles attributed to JNK1 and JNK2 remain unclear, therefore JNK as a target in this context needs to be investigated further using more selective tools.
Here we explore JNK1 and JNK2 function in breast cancer cell lines using lentiviral shRNA to selectively knock down these targets. In order for comparisons to be drawn, SP600125 will be used to ensure that previously published data can be reproduced in the MCF-7 cell line.
Blots were washed for 15 minutes in TBS-T before being incubated for 2 hours in 0.5% BSA (w:v) TBS-T with 1:7500 dilution of HRPconjugated secondary antibody. Blots were developed using ECL reagent followed by exposure to Kodak X-ray film. Antibodies:  For experiments with SP600125, MCF-7 cells were treated with a lower concentration of SP600125 (20 lmolÁL À1 ) due to repeated treatment after the initial measurement on day 2 and on days 4 and 6. Controls of media alone and 1% DMSO were also replenished.

| Cell viability
Cells were seeded onto 96 well plates and stimulated for 24 hours with either 1% DMSO or 20 nmolÁL À1 taxol. 10 lL of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) was added to each well and cells were incubated for 2 hours. Media were then removed from the wells and 100 lL of DMSO was added to dissolve the purple formazan product. Absorbance (570 nm) was then measured to estimate cell viability.

| Clonogenic assay
Cells were seeded onto 60 mm 2 dishes and incubated overnight at 37°C in a humidified air containing 5% CO 2 . Cells were starved for 24 hours and then exposed to 10 J/m 2 of UV radiation. Media were replaced with complete media and plates were incubated for 12-15 days, with media replenished on days 5 and 10. After incubation, the media were removed; cells were washed with PBS, fixed with methanol, and stained with Giemsa for 20 minutes. After stain was removed, formed colonies were counted manually and survival fractions were calculated. For SP600125 experiments, cells were pretreated with 30 lmolÁL À1 of SP600125 1 hour before exposure to UV radiation, the media remained on the cells 6 hours after exposure and were then replaced with complete media and followed the same course as previously described.

| Data analysis and processing
All statistics were calculated using GraphPad Prism version 5.01.
Datasets were analyzed for statistical significance using either a one or two-way analysis of variance (ANOVA). P values <.05 were con- These results coincide with those published by, 15 thus confirming reproducibility of this JNK phenomenon in MCF-7 cells.

| Generation of MCF-7 cell lines containing JNK individual isoform knockdown
After confirming that inhibition of JNK by SP600125 influenced cell cycle progression and proliferation, we next investigated which JNK isoforms played a role in these processes. The Lentiviral delivery method of shRNA has been used to knockdown JNK isoforms in different cell lines including human epithelial, 18 human liver cancer, 19 and mouse mammary tumor cells. 20 As this has allowed differences in isoform function to be determined in these studies, we used len-  Figure 2).

MCF-7 proliferation, cell cycle progression or phosphorylation of c-Jun
After cell lines were successfully generated and knockdown was confirmed, these cells were used to investigate the effects of JNK isoform knockdown on cell cycle progression using FACS analysis.  Figure 3A). Interestingly there were not any major differences observed between the cell lines, including the control and nontarget (NT) lines ( Figure 3A). There was also no cell cycle arrest produced by the knockdown of JNK demonstrating contrasting results when compared with the experiments using SP600125. Similarly, JNK isoform knockdown had little effect on the phosphorylation of cJun ( Figure 3B) and the proliferation of MCF-7 cells ( Figure 3C) where all cells produced a fold growth of between five and sevenfold with slight variation between experiments taken into consideration. Interestingly, when experiments using SP600125 were repeated in JNK isoform knockdown cells, loss of JNK expression did not alter the effects produced by SP600125 ( Figure S1).

| UV-induced cell death is independent of JNK in MCF-7 cells
Since the knockdown of JNK isoforms did not affect processes

| DISCUSSION
Extensive research has been carried out to demonstrate that the JNK pathway, and now more specifically the individual JNK isoforms themselves, play key roles in both cancer cell survival and cell death processes. 21 The outcome of this signaling is determined by a number of variables including cell type, 22 cell location, 23  also been reported to inhibit 13 other protein kinases, including AMPK, CDK2, and SGK. 9 Although JNK is an attractive target, there is yet to be a successful drug developed which can inhibit this protein without off-target effects. 8 In order for progress to be made in this area, more selective approaches need to be adopted to ensure that the effects observed are due to JNK and not one of the many off-targets inhibited by SP600125.
In our own investigation, we managed to recapitulate previously published findings using the SP600125 inhibitor. If the pharmacological data generated using SP600125 were to be solely relied upon, our data would similarly support a role for JNK signaling in MCF-7 cells proliferation and cell cycle progression. However the data presented in our study highlight the critical need of adopting a more selective approach when interrogating the JNK pathway and not relying solely on the use of SP600125.
In this study, stable and selective lentiviral knockdown of JNK1 and JNK2 was achieved in MCF-7 cells. Although consistent JNK isoform knockdown was achieved, no effect on cell death induced by UV radiation or taxol was observed. This was confirmed using a variety of approaches including cell viability assays, Western blotting, and FACS analysis. These results were surprising given that previous studies using SP600125 had proposed that JNK play a role in both UV 15,27 and taxol- 28  F I G U R E 4 UV-induced cell death is independent of JNK. (A) Cells were starved for 24 hours, exposed to 30 J/m 2 of UV radiation for 24 hours and then samples were collected and expression levels of cleaved PARP, pJNK, JNK1, JNK2, and GAPDH were analyzed. (B) Cells were starved for 24 hours, exposed to 10 J/m 2 UV radiation, cells were replenished with complete media and incubated for 12-15 days. A clonogenic assay was used to measure successful colonies formed for both exposed and unexposed cells. Data represent mean AE SEM of 3 independent experiments JNK signaling has been linked to both proliferation 17  Trying to determine JNK isoform function in cancer is very challenging due to the differences in JNK function between cancer types, 19,32 however, with more selective tools becoming available it should be possible. Studies using JNK knockout animals clearly present a good rationale for why targeting JNK isoforms may be of therapeutic value, 10 however firm translation of these findings into human cell models is crucial. For example, JNK1 but not JNK2 was shown to be required for UV-mediated death in MEFs. 33 However, in this study JNK knockdown had no effect on MCF-7 cell death induced by UV exposure. Differences in the translation of these events from mouse cells to human cell models may account for these conflicting findings. The use of JNK knockdown, or gene deletion approaches such as CRISPR, in other cell systems may help to shed some light on the role for JNK in human cell growth and death.
What is clear from our study is that any interpretations derived from studies that rely solely on the use of SP600125 need to be treated with caution when attributing the JNK pathway to MCF-7 cell growth and death. Due to the sheer number of other serine/ threonine kinases inhibited by SP600125, it would be wise to employ more selective ways to validate the role of JNK in cancer cell function. While shRNA approaches have their own set of challenges to ensure complete knock down of protein levels are achieved, the availability of gene editing approaches such as CRISPR will pave the way for a better fundamental understanding of the role of JNK (or any other target) in human cancer cell function. This will be essential if progress in the field is to be made and new targets are to be identified for anticancer therapies.

ACKNOWLEDG EMENTS
The study was supported by the University of Strathclyde. RW was funded by a University of Strathclyde funded PhD Studentship. MC is a University of Strathclyde Chancellor's Fellow and AMS springboard recipient (SBF001\1009).

DISCLOSURES
The authors have no conflict of interest to declare.

AUTHOR CONTRI BUTIONS
Rachel Wood: conceived the project, carried out the majority of the experimental work and wrote the manuscript. Robin Plevin: con- In contrast shRNA knockdown of JNK inhibited the phosphorylation of JNK however this had no effect on cell cycle progression or proliferation of MCF-7 cells. SP600125 has been demonstrated to inhibited kinases CDK2 and SGK 9 both of which have been shown to play a role in breast cancer cell proliferation and cell cycle progression 29,30 and therefore this could be an alternative path as to which SP600125 can elicit the effects produced in this study