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Silencing of the CSNK2β gene by siRNA inhibits invasiveness and growth of MDA-MB-231 cells

Background Breast cancer is most common cancer and accounts for one-fourth of all cancer diagnoses worldwide. Treatment of triple-negative breast cancer is major challenge and identification of specific drivers is required for targeted therapies. The aim of our present study is to elucidate the therapeutic potential of CSNK2ß silencing in triple negative breast cancer MDA-MB-231 cell. Methods CSNK2ß gene has been knockdown using siRNA and silencing was estimated by both real time and western blot. Cell Titer-Glo (CTG) and colony formation assay and wound healing assay, cell cycle analysis by flow cytometry was performed to assess the role of CSNK2ß in cell proliferation, migration, cell cycle, and oncogenesis. Morphological assessment of nuclear condensation, apoptosis by Hoechst staining and measurement of intracellular ROS production was examined using fluorescence microscopy. Real time PCR and western blot was done to study the expression of genes related to cell proliferation, survival, metastasis, apoptosis, and autophagy. Results Silencing of CSNK2ß in MDA-MB-231 cells resulted in decreased cell viability, colony formation, and migratory potential. Cell cycle analysis showed that growth inhibitory effect was mediated by arresting the cells in G2/M phase. Furthermore, we demonstrated that silencing of CSNK2ß increased the nuclear condensation and intracellular ROS production. CSNK2ß regulates the expression of BAX, Bcl-xL, caspase 3, Beclin-1, LC3-I, p-ERK, p38-α, c-Myc, MAPK, c-Jun, NF-ĸB, β-catenin, E2F1, PCNA. We have also shown the functional relationship between CSNK2ß, PIN1, and PTOV1 by western blotting. We have first time reported that silencing CSNK2ß using siRNA can inhibit invasiveness and proliferation of MDA-MB-213 cells. Conclusion Our results suggested that CSNK2ß silencing may offer future therapeutic target in triple negative breast cancer.

In a mammalian cell, CSNK2β is phosphorylated at Ser 209 at its autophosphorylation site and at Ser 53B, in a cell cycle-dependent manner and in vitro by p34 cdc5 [14,15].
To understand its physiological importance in the regulation of multiple candidate target proteins, we focused our study on the role of CSNK2β in the tumorigenesis of human breast cancer (MDA-MB-231 cell) in vitro. In the present study, we used the RNA interference strategy to knockdown the CSNK2β gene and study the gross oncogenic activity in an in-vitro cell-based system. We evaluated its clonogenic, invasive, proliferative and apoptotic properties in MDA-MB-231 cells using siRNA. We found that CSNK2β regulates the cell proliferation by targeting NF-κB, Wnt, JNK and MAPK pathway proteins and also modulates the expression of PIN1 and PTOV1 oncogene.
Our findings suggest that CSNK2β can be used as a novel target for breast cancer therapy.

Cell culture
MDA-MB-231 cell was purchased from National Centre for Cell Science (Pune, India).
The cells were cultured in L-15 medium supplemented with 10% FBS, penicillin (100 unit/ml) and streptomycin (100µg/ml). The cell culture was maintained at 37°C in humidified air containing 5% CO 2 .

Transfection
Cells were cultured in 6 well plates one day before siRNA transfection. We used 25 nanomolar of each siRNA and made complex in Opti-MEM media. Similarly, the complex of Lipofectamine RNAiMAX (4 µl/each well) and Opti-MEM was made and incubated for 5 minutes at room temperature. After that, both the complexes were mixed in 1:1 proportion and incubated for 25 minutes at room temperature. Cells were treated with Opti-MEM-siRNA Lipofectamine complex and incubated at 37°C for 72 hours. The sequences used in siRNA and the target sequence for the genes in our study are

Construction of CSNK2β Overexpression plasmid and transfection
CSNK2β was cloned into pcDNA 3.1(+) expression vector. 2.5 µg of empty vector and Overexpression clone was used for transfection. Briefly , 1:1 complex of lipofectamine   2000 plus Opti-Mem and plasmid plus Opti-Mem were made and incubated for 20 minutes. The cell plating number was similar to siRNA transfection.

Cell viability assay
Cell viability was accessed with CTG assay (Promega, Madison, WI). Briefly, MDA-MB-231 cells were seeded in 96 well white culture plate at a density of 1000 cells per 180 µl of medium per well with 20 µl of siRNA complexes for CSNK2β and incubated at 37°C, 5% CO 2 and incubated for 24 hours. On the next day, the media containing the complex was changed with the fresh media and further incubated till 96 hours. The cells were treated in quadruplets with respective siRNA. The reagents were prepared according to manufacturer's protocol. After incubation 100 µl of fresh media was added to each well followed by 100 µl of reagent and kept on a shaker for 2 minutes to induce the cell lysis.
The plate was incubated for 10 minutes at room temperature to stabilize the luminescence signal. Luminescence was measured using microplate ELISA reader (Bio Tek, Winooski, Vermont, US).

COLONY FORMATION ASSAY
MDA-MB-231 cells were transfected with Scramble and CSNK2β siRNA and incubated for 48 hours. After the cells were trypsinized, collected and counted.1000 cells /pell were taken from each Scramble and Csnk2β transfected samples and seeded in 6 well plate. The cells were allowed to grow for 3 weeks until 80-90% confluence was observed. Then the cells were washed with DPBS, fixed with 3.7% formaldehyde for 30 min and stained with 0.4% crystal violet. The cells washed DPBS for 2-3 times and allowed to dry. The colonies were counted using Image J software. to PVDF membrane (100 volts for 1 hour). The membranes were blocked using 5% skim milk in Tris buffer saline with 0.1% tween20 (TBST) for 2 hours at room temperature. The blots were incubated with primary antibodies overnight at 4°c on a rocker followed by incubation with HRP conjugated secondary antibodies. The blots were developed using enhanced chemiluminescence (ECL, Biorad). β actin was developed as a loading control.

RNA extraction, cDNA preparation, and real time PCR
Cells were transfected for 72 hours with the respective siRNAs and RNA was extracted using TRIzol lysis reagent using manufacturers protocol. 2µg of RNA was taken and pretreated with DNase followed by cDNA synthesis with oligoDT primers. The thermocycler condition was 65° c, 5 min, 42°c, 60 min and 72°c (final extension). Real time PCR was done with iTaq SYBR green mix (Biorad) using ABI 700,(Invitrogen).
PCR was carried out at following conditions.

Hoechst staining
1.5 10 5 cells were seeded in 6 well plates. On the next day, the cells were transfected with Scramble and CSNK2β siRNA and incubated for 72 hours. Then the cells were stained with Hoechst 33342 (2µg) stain for 15 minutes in dark. Cells were once washed with DPBS and the images were taken using Nikon fluorescent microscope.

Measurement of the intracellular ROS production
Intracellular ROS generation was accessed by fluoroprobe DCF-DA (Invitrogen).

Statistical Analysis
Data were represented as a mean ± standard deviation. The level of significance between two groups was calculated by t-test. P < 0.05 was considered statistically significant.

Expression of CSNK2β is altered in a multitude of tumor types.
cBioPortal data shows that CSNK2β has alterations (mutation, amplification, and deep deletion) across the human cancer types. The histogram shows the potential cancer relevance of CSNK2β and high frequency of alteration in Breast (BCCRC xenograft) that prompted us to validate its function in breast cancer ( Figure 1) [28].

Silencing of CSNK2β inhibits the colony formation of MDA-MB-231 cells
We also performed the clonogenic assay which associates with the tumor formation in vivo [29]. Cells were transfected with Scramble and CSNK2β siRNA in 6 well plates and

Silencing of CSNK2β induces chromatin condensation indicating apoptotic morphology
After 72 hours post-transfection with CSNK2β siRNA remarkable changes such as chromatin condensation and nuclear fragmentation was seen in the test samples compared to Scramble using Hoechst 33258 staining ( Figure 5A). This morphological examination interprets that CSNK2β has a profound role in cell survival and silencing it drives the cells towards apoptosis.

Silencing of CSNK2β increases the intracellular ROS production thereby enhancing cell death
ROS possess double edge sword property both as oncogenic, maintaining sustained and increased proliferation of cancer cells as well as tumor suppressor leading to cell death when the aberrant increase in intracellular ROS arises due to any kind of stress [30]. The fine distinction between the ROS activity and the fate of the cells depend on the balance between pro-oxidant and antioxidant or redox status of the cells. It is a good idea to find oxidative stress modulators as an anti-cancer strategy [31]. In our experiment, we found the increased production of ROS in CSNK2β siRNA transfected cells compared to scramble siRNA transfected cells ( Figure 5B). This data suggested the CSNK2β modulates the intracellular ROS production in MDA-MB-231 cells.

Silencing of CSNK2β induces the cell death of MDA-MB-231 cells by both apoptosis and autophagy mechanisms.
To unravel the cell death mechanism induced after the knockdown of CSNK2β, western blot analysis was performed to determine the level of proteins related to apoptosis after 72 hours of post-transfection. We found that there was increased expression of BAX and repair) and E2F1 (transcription factor) ( Figure 7F). Silencing of CSNK2β abrogated the expression of NF-κB which is constitutively expressed in breast tumors ( Figure 7G) [33]. We also studied the expression of integrin 1 beta, a heterodimeric receptor that senses both external and internal cues and has an important role in tumor biology.
CSNK2β siRNA inhibits the expression of Integrin 1β thus decreasing proliferation and invasion (Figure7 H).

PTOV1.
From our previous study, we found that there was the functional relationship between PIN1, PTOV1, and CSNK2β. We have shown that all the three genes share the functional relationship through c-JUN expression that mediated the cancer progression in prostate cancer (PC3 cells). We proposed that these genes fall in the same pathway and mediates their function either by direct interaction with PIN1 or indirectly by promoting the oncogenesis of PIN1 [34]. So we were interested to find the relationship

Discussion
Breast cancer is the most common cancer occurring in women of both developed as well as developing countries. It was estimated that 1.7 million new cases were diagnosed in 2012 which was 12% of all new cases and 25% of all cancers in women (https://www.wcrf.org/int/cancer-facts-figures/data-specific-cancers/breast-cancer statistics). Mortality due to breast cancer has been decreasing due to early diagnosis, improved adjuvant therapy, low usage of hormone replacement therapy [35,36].
Despite all these advancements and outcomes, triple negative breast cancer still remains a major challenge. Statistics show that TNBC represents 10-15% of all breast cancer and has a poor prognosis compared to other subtypes of cancer (ER, PR, and HER2/Neu) [37]. Thus it is indispensable to explore the potential candidate target to reduce the rate of metastases, morbidity, and mortality due to breast cancer and Controlled production of ROS is beneficial for proliferation and viability of cancer cells but its elevated level may be genotoxic and cause apoptosis of cancer cells [30,31].
Microscopic study of CSNK2β silenced MDA-MB-231 cells showed the increase in ROS production and nuclear condensation. Both the features support that the cells were in the process of apoptosis after the silencing with CSNK2β siRNA. Studies on different isoforms of casein kinase and their subunits in eukaryotic biology and cancer have been studied [1][2][3], but the possible mechanistic study of CSNK2β in cancer was unclear.
Although CSNK2β is a regulatory subunit of CSNK, it is highly conserved protein and possesses independent function. Thus we anticipate that finding the new molecules within the CSNK2β regulatory network will tease out its role in tumorigenesis. To get the further insight into the biological significance of this protein we performed the expression study of the key molecules related to cell proliferation, survival, cell cycle, apoptosis and autophagy-related genes by western blotting and real time PCR.
Although the improved chemotherapy and use of adjuvants have decreased death rates due to breast cancer, understanding the response to treatment and apoptosis is still a major concern [38]. Here we have shown the induction of BAX (proapoptotic), suppression of Bcl-xL( antiapoptotic), activation of procaspase 3 with an elevated level of cleaved caspase 3 in CSNK2β siRNA transfected sample suggesting the cell death via apoptosis. Paradoxically autophagy is utilized as a survival mechanism by tumor; it can also promote the caspase-independent form of cell death and can be used as a cancer treatment modality [39,40]. Moreover, BECN1 and LC3 which are autophagy markers were increased in CSNK2β knockdown samples. Since it follows both apoptosis and autophagy cell death mechanism, CSNK2β is an interesting molecule for targeted cancer therapy.
Raf/MEK/ERK pathways are activated in many tumors (prostate, breast, leukemia, melanoma, thyroid) which transmit the signals from cell surface receptors to transcription factors and can be exploited for therapeutic intervention [41,42]. In the present study, we found that CSNK2β regulates the expression of P-ERK, p38-α, c-MYC, MAPK8 proteins which are connected to MAPK pathway in MDA-MB-231 cells.
These data suggested that targeting CSNK2β might be a potential strategy to improve clinical outcomes in future. Numbers of studies have been carried out on the dysregulation of Wnt/β-catenin in human breast cancer and is good clinical and pathological marker with the poor survival outcome [43,44]. Consistent with these reports, our data also showed that knockdown of CSNK2β down-regulates the expression β -.catenin. PCNA is earlier reported as a reliable marker to access the growth and predicting the prognosis in breast cancer [45]. Thus we investigated the effect of silencing of CSNK2β on PCNA and its downregulation supported the previous findings. A recent study on the interbreeding of MMTV-PyMT mice with E2F1, E2F2, or E2F3 knockout mice showed that in addition to cell cycle control E2F targets the number of genes related to angiogenesis, extracellular matrix modification, proliferation and survival of tumor cells which was important for metastasis [46]. We also found that CSNK2β reduces the expression of E2F1 which might affect a large fraction of genes in breast cancer. NF-κB expression leads to the induction of genes related to apoptosis, cell cycle, cell invasion which contributes to tumorigenesis, chemoresistance and radioresistance [47]. Our result showed that CSNK2β might regulate the cell proliferation through the NF-κB pathway. From wound healing experiment we deduce that CSNK2β has a remarkable role in cell migration. This was further supported by the decrease in integrin 1B, glycoprotein receptors that mediate anchorage and migration of cells via cell matrix and cell-cell interactions [48].  [49,50].
In continuation of our previous study [34], we have shown that overexpression of CSNK2β increases the expression of PIN1 while silencing it decreases its expression.
Although the study on PIN1 and CSNK2α is reported previously our results provided a new avenue for further exploration. Also, we have shown that Overexpression of PIN1 and CSNK2β elevated the level of C-Jun and Knockdown by siRNA reduced the expression. There are several reports of PTOV1 and human breast cancer.
Overexpression of PTOV1 promotes tumor progression and is a predictor of poor prognosis in breast cancer [51]. Also, PTOV1 promotes the tumorigenicity by activating Wnt/β-catenin pathway in breast cancer [52]. In our study, we have shown an initial report that overexpression and knockdown of CSNK2β both increased and decreased the expression of PTOV1respectively.Our previous study on co-immunoprecipitation showed that PIN1 interacts with PTOV1. These results support our previous finding that PIN1, PTOV1 and CSNK2β fall in the same pathway and either directly interact or cooperatively promote the oncogenesis [34].
This conjecture provides a new scope for further experiments and validations to establish the relationship between these molecules and decipher their role in cell proliferation, survival, migration, and cell death in breast cancer as well as other cancer models.

Conclusion
In summary, our findings from our laboratory have shown that silencing of CSNK2β with siRNA significantly reduced the cell viability, colony formation potential, cell migration