BRD9 controls Oxytocin signal pathway in gastric cancer via CACNA2D4 and CALML6

Background: First-line chemotherapeutic agents have great activation treatment in cancers, but the side effect of these kind drugs also hurt the healthy cells, in some cases, cancer cells are induced drug resistance to chemotherapeutic agents. The molecular mechanisms underlying such side effect have been studied in a range of cancer types, yet little is known in depressing the adverse effect of chemotherapeutic drugs by targeting BRD9 in gastric cancers. Methods: We used two gastric cancer cell lines (MGC-803 and AGS) for comparison. We applied molecular and cellular techniques to measure cell survival, mRNA expression, investigated clinical data in the consensus of The Cancer Genome Atlas, and combined with the High-Throughput Sequencing approach in MGC-803 cells and AGS cells, for global gene expression analysis in inhibiting BRD9 conditions. Results: Our studies have shown that cancer cells with BRD9 over-expression, MGC-803 cells, are more sensitive to BRD9 inhibitors, BI9564 or BI7273, than AGS cells. Its mechanism is related to the regulation of CACNA2D4 and CALML6 oncogenes in the Oxytocin signaling pathway. And BRD9 inhibitors can enhance the sensitivity of gastric cancer MGC-803 cells to adriamycin and cisplatin, so we may reduce the dosage of chemotherapeutic agents in curing gastric cancers with BRD9 over-expression by combining BI9564 or BI7273 with adriamycin or cisplatin. Conclusions: Our study elucidates the feasibility and effectiveness of inhibiting BRD9 to reduce the adverse effect of first-line chemotherapeutic agents in treating over-expressed BRD9 gastric cancer, providing a scientific theoretical basis on chemotherapy regimen in over-expressed BRD9 gastric cancer. real-time PCR MGC–803 BI9564 BI7273 treatment; The RNA expression levels of CALML6 measured by quantitative real-time PCR in MGC–803 cells after BI9564 and BI7273 by finding exact oncogenes targeted BRD9, tested some mRNA in oxytocin pathway after added BRD9 inhibitors, found the gene CACNA2D4 CALML6 are the by BRD9 over-expressed gastric cancer, and elucidate the feasibility and effectiveness of combining BRD9 with first-line chemotherapeutic agents in curing BRD9

Producing adverse effects in curing cancer patients is a main problem in both novel targeted therapeutics and conventional chemotherapeutics [1]. Adriamycin (ADR, Doxorubicin, Dox) and cisplatin (CDDP), first-line chemotherapeutic medicines, are potent chemotherapeutic agents which are used for the treatment of numerous cancers [2,3].
Adriamycin's mechanism of effect involves inhibiting the synthesis of DNA and damage to DNA [4], and the formation of reactive oxygen species (ROS) [5] to create oxidative stress in the cellular environment. The mechanism of cisplatin (CDDP) is related to DNA Doublestranded covalent crosslinking and forming DAN-cisplatin adducts [6]. Although the action mechanism of adriamycin and cisplatin are multiple, many healthy cells are hurt to some extent and some cancers are developing drug resistance to them too [6]. Damaged healthy cells in cancer patients will depress their immunity, bad for their recovering healthy. Many cancer patients congenital or acquired drug resistance to chemotherapy including ADR and CDDP. The ways of drug resistance of cancer cells include the increased expression of DNA repair genes, abnormal drug transport pathway, acetylation of histones, epigenetic modifications activating drug resistance pathway and so on [7,8]. these phenomena seriously reduce the efficacy and anti-cancer spectrum of first-line chemotherapeutic drugs. So, the most urgent and important problem in the clinical application of first-line chemotherapeutic drugs is downregulation these kinds of side effects.
Recently many studies showed abnormal epigenetic control is one of the important reasons in causing carcinoma's drug side effects. In many carcinoma's types, epigenetic regulation involving gene expression, DNA repair, and DNA replication [9,10]. Epigenetic regulation protagonists include writers, readers, and erasers, as well as members of chromatin-remodeling complexes, mutations in these genes, are pervasive in cancer, and few if any cancers escape mutations in one of these major chromatin rheostat proteins. Some studies showed that members of the mammalian SWI/SNF chromatin-remodeling complex are mutated in more than 20% of all cancers [11]. These mutations and alongside mutations in histones themselves can promote the development of malignancy and resistance to drugs in cancer cells [12]. These findings firmly establish that epigenetic dysregulation plays a causal role in cancer initiation, progression and developing drug resistance. On the other hand, the drugs targeting epigenetic regulator have fewer side effects than chemotherapeutic drugs' in healthy cells. Because most chemotherapeutic agents targeting oncogene DNA, many healthy cells' DNA may be hurt by chemotherapeutic agents either.
As epigenetic readers, bromodomain proteins can recognize acetylated histone tails to facilitate the transcription of target genes. Based on structural conservation the human bromodomains, approximately 60 as we knew, can be divided into eight sub-families. The family IV of bromodomain-containing proteins include seven members (BRPF1, BRPF2, BRPF3, BRD7, BRD9, ATAD2, and ATAD2b) [13]. While the bromodomains of BRD7 and BRD9 are members of the SWItch/Sucrose Non-Fermenting (SWI/SNF) chromatin remodeling complex, which regulates gene expression, as showed in figure 1A. BRD9 has been shown to recognize the doubly acetylated histone H4K5acK8a, the di-propionylated ligand H4K5prK8pr ( Figure 1B, Figure 1C) and histone H4K5buK8bu [14].
We selected gene BRD9 through consensus of The Cancer Genome Atlas (TCGA). In clinical cases and cancer cells, mutations of BRD9 are common, Cell lines data from TCGA database shows 21% of cancer cells mutate in BRD9 ( Figure 1D). Some study shows that BRD9 abnormal expression is related to cervical cancer, non-small cell lung cancer and liver cancer [15][16][17]. This kind of mutation is also found in endometrial cancer, squamous cell lung cancer and prostate adenocarcinoma [18]. One study showed that PC9 cell mutated in BRD9 was drug resistance to EGFP inhibitor [19]. We can see that gene BRD9 mutation is common, and up to now none study investigated the role of BRD9 in gastric cancer.
Data from the TCGA database shows 26% mutation was found in gastric cancer ( Figure   2A). Terry D. Crawford's study showed that BRD9 inhibitor decreased BRD9 binding to chromatin, and prevented the emergence of a drug-resistant population in EGFR mutant PC9 cells treated with EGFR inhibitors [19]. Anja F. Hohmann and his team found that the BRD9 and the SWI/SNF chromatin remodeling complex are hyperactive in acute myeloid leukemia (AML) cells, they sustain MYC transcription, rapid cell proliferation, and a block in differentiation. Inhibiting BRD9 can reverse the proliferation of cancer cells induced by SWI/SNF [20]. Notwithstanding SWI/SNF composition and BRD9 were studied in these two kind cancer cells, the role of BRD9 remains to be studied in gastric cancer.
The incidence of gastric cancer is highest in Eastern Asia [21], to find the effective way for BRD9 over-expressed gastric cancer treatment, it is necessary to study the role of BRD9 in this kind of cancer. Here we show that BRD9 promotes CACNA2D4 and CALML6 expression in the Oxytocin signal pathway and induces gastric cancer cells proliferation. AGS cells and MGC-803 cells are two type of gastric cancer cells, the expression of BRD9 in MGC-803 cells is higher than AGS cells. MGC-803 cells are more sensitive to BRD9 inhibitors (BI9564 and BI7273) than AGS cells. The results described here reveals the potential signaling pathway controlled by BRD9 in BRD9 over-expressed gastric cancer contexts.
And we found that when we combining BI9564 or BI7273 with adriamycin or cisplatin to treat these two types gastric cancer cells the dosage of adriamycin or cisplatin needed by MGC-803 cells was downregulated greatly. The data and analyses provide the feasibility and effectiveness of inhibiting BRD9 to reduce the adverse effect of first-line chemotherapeutic agents in treating BRD9 over-expressed gastric cancer, providing a scientific theoretical basis on chemotherapy regimen in gastric cancer. Selleck.) plates were added to standard tissue incubator conditions. Medium was removed and cells were solubilized in 150 μl DMSO. The intensity of formazan was measured at 490 nm using an automated microplate spectrophotometer (iMark; BioRad, Hercules, CA, USA).
The viability was calculated as (OD value of the treated group/OD value of untreated group) x 100%. We performed three times in every experiment.

Analysis of drug sensitivity activity
The viability of AGS and MGC-803 cells following treatment with ADR or CDDP in the presence or absence of BI9564 or BI7273 (500nm or 1000nm) were analyzed by MTT assay. Following plotting of the dose-response curve, the IC50, the concentration of drug inhibiting 50% of cells was calculated.
Quantitative real-time PCR After dealing as indicated, total mRNA of cells was extracted with TRIZOL reagent. The first strand of cDNA synthesis was generated from 2 μg total RNA using oligo-dT primer    Figure 4C); we also screened 12 downregulation genes and 11 upregulation genes in AGS cells after inhibited BRD9 ( Figure 4D). On the basis of figure   4C and figure 4D, we used software, database for annotation, visualization and integrated discovery-DAVID, to analysis these changed genes and find some high score carcinogenic pathways ( Figure 4E). Then we searched a large number of references, we found one of these pathways, the Oxytocin signaling pathway, may be related to BRD9 inhibitors, and its mechanism with BRD9 is remaining to be studied.

BRD9 mediated CACNA2D4 and CALML6 expression
To find the exact genes controlled by BRD9 in the Oxytocin pathway, we applied the KEGG data base, chose some changed genes, and also verify the chose genes with RT-PCR assay. From KEGG we found that upregulated CACNA2D4 will promote CALML6 to raise, these effects will induce anti-apoptotic and inhibit oxytocin production ( Figure 5A). The RT-PCR assay showed that the expression of CACNA2D4 and CALML6 were all downregulated after treated by BI9564 or BI7273 ( Figure 5B and 5C), that is to say, inhibiting BRD9 can induce apoptotic and prompt secretion of oxytocin. Studies have shown that oxytocin secretion is negatively correlated with the risk of esophageal, gastric and pancreatic cancer [23][24][25][26][27]. Breastfeeding can induce production of oxytocin, and in this way, the risk of esophageal cancer reduced by 54% [28]. Therefore, promoting the secretion of oxytocin is conducive to inhibiting the occurrence and development of gastric cancer. From above results we speculate that BRD9 regulates the oxytocin signaling pathway in gastric cancer, and is associated with carcinogenic genes CACNA2D4 and CALML6.  Table 1), and when the dosage of BI9564 was enhanced to 1000nm and added with 2.5ug/ml ADR in MGC-803 cells the cell viability was cut down to 15.95% ( Figure 6D, Table 1). On the contrary, when 5ug/ml ADR added into MGC-803 cells, the cell viability still high at 41.48% ( Figure 6A, Table 1), and when the ADR dosage increased to 10g/ml the cell viability is 35.6% ( Figure 6A, Table 1). The viability values of single using ADR to kill MGC-803 cells are higher than combination method, even if the dosage of ADR as high as 10g/ml. From these results, we can see that combination BI9564 with ADR in MGC-803 cells will greatly reduce the MGC-803 cell viability, and the combination medicines effect is more excellent than only treated with ADR in MGC-803 cells. Then we change the BRD9 inhibitor BI9564 to BI7273, the same trends were shown in table 2 and Figure 6E, 6F. The effects of combination BI7273 with ADR in MGC-803 cells is better than only treated with ADR. To test the university of these methods in curing over-expressed BRD9 gastric cancer patients, we changed ADR to CDDP in these experiments. From table 3 and table 4 we get the same conclusion that combination BI9564 or BI7273 with CDDP will greatly cut down the survival rate in over-expressed BRD9 gastric cancer cells ( Figure 6F, 6G, 6H, 6J).
These results shown that BI9564 and BI7273 can enhance the sensitivity of over-    Adriamycin (ADR, a chemotherapeutic drug) and cisplatin (CDDP, a chemotherapeutic drug) are first-line chemotherapeutic agents for treating solid tumors [29]. But a large number of patients induced great side effect after chemotherapy and produce successive tumor reversion and made the failure in treating cancer with ADR or CDDP [30][31][32][33]. In our study, we found BRD9 over-expressed condition in gastric cancer is universal, the clinical incidence rate is 26%. We used experiment to verify the function of BRD9 in cancer cells and found that if we use BRD9 inhibitors to treat MGC-803 cells and AGS cells the overexpressed BRD9 MGC-803 cells were more sensitive to BI9564 or BI7273 than AGS cells.
To examine the potential mechanism may mediate by BRD9 in this phenomenon, we performed a screening for changed genes towards BRD9 inhibitors treatment, and with the help of analysis software DAVID, we found the BRD9 may be related with cell adhesion molecules, oxytocin signal pathway, and gastric acid secretion and so on. What is the special pathway in the BRD9 over-expressed gastric cancer controlled by BRD9? With this question we resorted to some references, some studies have shown that oxytocin secretion is negatively correlated with the risk of esophageal, gastric and pancreatic cancer [24][25][26][27][28], so we thought that oxytocin signal pathway may play a key role in BRD9induced drug resistance in MGC-803 cells.
The above conclusion is based on the software prediction, the truth conclusion should be