Inhibition of TRIM32 by ibr‐7 treatment sensitizes pancreatic cancer cells to gemcitabine via mTOR/p70S6K pathway

Abstract Pancreatic cancer is one of the most notorious diseases for being asymptomatic at early stage and high mortality rate thereafter. However, either chemotherapy or targeted therapy has rarely achieved success in recent clinical trials for pancreatic cancer. Novel therapeutic regimens or agents are urgently in need. Ibr‐7 is a novel derivative of ibrutinib, displaying superior antitumour activity in pancreatic cancer cells than ibrutinib. In vitro studies showed that ibr‐7 greatly inhibited the proliferation of BxPC‐3, SW1990, CFPAC‐1 and AsPC‐1 cells via the induction of mitochondrial‐mediated apoptosis and substantial suppression of mTOR/p70S6K pathway. Moreover, ibr‐7 was able to sensitize pancreatic cancer cells to gemcitabine through the efficient repression of TRIM32, which was positively correlated with the proliferation and invasiveness of pancreatic cancer cells. Additionally, knockdown of TRIM32 diminished mTOR/p70S6K activity in pancreatic cancer cells, indicating a positive feedback loop between TRIM32 and mTOR/p70S6K pathway. To conclude, this work preliminarily explored the role of TRIM32 in the malignant properties of pancreatic cancer cells and evaluated the possibility of targeting TRIM32 to enhance effectiveness of gemcitabine, thereby providing a novel therapeutic target for pancreatic cancer.


| INTRODUC TI ON
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer, and is also notorious for its high malignancy and mortality after diagnosis. 1,2 Since most PDAC patients are diagnosed at late stage, systemic chemotherapy and targeted therapy are considered as important intervention strategies for PDAC patients. 3,4 Although tremendous efforts have been devoted to explore efficacious compounds for treating PDAC, there are still few regimens that have achieved desired outcomes in clinical studies. 5 It is assumed that the hyperactivation of both MEK/ERK and AKT/mTOR pathways in approximately 90% of PDAC patients accounts for their insensitivity to cancer drugs. [6][7][8] Inhibition of either pathway would be insufficient to restrain the proliferation of PDAC cells, except for those possess unique mutant aberrations that could be targeted by specific agents. 9,10 On the contrary, although gemcitabine remains as the first-line chemotherapy in PDAC treatment for decades, strategies to improve the efficacy of gemcitabine are facing numerous obstacles and the results are still challenging. [11][12][13] Therefore, to discover novel agents that could efficiently inhibit the proliferation of PDAC or investigate key components that play decisive roles in gemcitabine sensitivity is extremely compelling in PDAC therapy.
Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase, thus stalling the development and maturation of B cells. 14,15 In pancreatic adenocarcinoma, ibrutinib was found to exert potent antitumour activities in xenograft models. 16 Unfortunately, ibrutinib failed to corroborate the efficacy of nab-paclitaxel plus gemcitabine in RESOLVE trial (PCYC-1137) last year, which put the applications of ibrutinib for PDAC into a dilemma. In our previously published work, where the structure of ibr-7 was referred, we have described ibr-7 as a novel derivative of ibrutinib. Ibr-7 exhibited its superior antitumour activity than ibrutinib via sufficient suppression of EGFR signalling pathway in non-small cell lung cancer cells. 17 In our recent work, we have found that ibr-7 was capable to increase the radiation-induced cell death by enhancing substantial DNA damage in PANC-1 and Capan2 cells. 18 However, the underlying mechanisms of ibr-7 in PDAC cells, and more importantly, whether ibr-7 could enhance the effectiveness of gemcitabine remain unclear. In another aspect, although previous reports have demonstrated the involvement of TRIM32 in progression of many types of malignant tumours, 19,20 its role in PDAC is yet fully investigated. In this study, we attend to fully explore the inhibitory effects and mechanisms of ibr-7 and meanwhile validate the potential role of targeting TRIM32 to enhance the efficacy of gemcitabine in PDAC cells.

| Apoptosis assay
Cells were seeded in 6-well plates (2 × 10 5 /well) and cultured overnight in a 5% CO 2 atmosphere at 37℃. After treatment with ibrutinib, Assays were performed on three independent experiments.

| Western blot Analysis
After treated with different concentrations of compounds, total proteins were extracted using RIPA lysis buffer. A total amount of 40 μg proteins were subjected to 12% SDS-PAGE and transferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated with primary antibodies overnight at 4℃. After washing with Tris-buffered saline with Tween 20, membranes were incubated with secondary antibodies at room temperature for another 1 h. The protein bands were visualized by adding ECL system WBKLS0050 (EMD Millipore, Billerica, MA, USA) and analysed using Bio-Rad Laboratories Quantity One software (Bio-Rad, Hercules, CA, USA).

| DAPI stain
BxPC-3 and SW1990 cells (8 × 10 4 cells/well) were cultured in 24well plates. After exposure to ibr-7, gemcitabine or the combination, cells were fixed with 4% paraformaldehyde for 20 min and stained with DAPI for 15 min. After washing with PBS, cells were observed under a fluorescence microscope (Nikon, Ti-E, Japan).

| mRNA library construction and sequencing
Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and
Cells were then transfected with the siRNA using jetPRIME

| Virus production and transfection
The 293T cells were seeded into 100 mm × 20 mm dishes. Until they

| Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells with TRIzol, precipitated with isopropyl alcohol and rinsed with 70% ethanol. Single-strand cDNA was prepared from the purified RNA using oligo (dT) priming (Invitrogen, Thermofisher scientific, Waltham, MA, US), followed by SYBR-Green (Qiagen, Hilden, Germany) and carried out using

| Transwell migration assay
Cell migration assays were performed using 24-well Transwells

| Colony formation assay
BxPC-3 cells were seeded into 6-well plates at a density of 1x10 3 /well and incubated for 24 h. The cells were then incubated with siTRIM32 or siControl. Following 24 h of treatment, the supernatant was removed and cells were cultured for a further two weeks.
Then, the cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa solution for 15 min at room temperature.

| Statistical analysis
The results are expressed as the mean ± SD of at least three independent experiments. Differences between means were analysed using Student's t test and were considered statistically significant

| Ibr-7 inhibited the proliferation of PDAC cells via suppression of mTOR and ERK phosphorylation
The anti-proliferation effects of ibr-7 or ibrutinib were determined in PDAC cells by using CCK-8 assays. In four tested PDAC cell lines, ibr-7 showed stronger anti-proliferation activity than ibrutinib ( Figure 1A), and there was a 10 fold to 60-fold difference between ibr-7 and ibrutinib in IC50 values (Table 1). Aiming to explore the underlying mechanisms of ibr-7, we examined essential proteins involved in regulating proliferation in three pancreatic cancer cell lines. As a result, 8 μM of ibr-7 effectively suppressed phosphorylated mTOR, p70S6 and S6, which were slightly influenced by ibrutinib or gemcitabine treatment ( Figure 1B). Besides, phosphorylated ERK was remarkably suppressed after treatment with 8 μM of ibr-7 or ibrutinib in BxPC-3, SW1990 and CFPAC-1 cells. Therefore, the dual-inhibitory effect of ibr-7 on both mTOR/ p70S6K and ERK might contribute to its potent anti-proliferation activity in PDAC cells.

| Ibr-7 induced mitochondrion-mediated apoptosis in PDAC cells
To Noticeably, apparent elevated expression of Bax was only seen after ibr-7 exposure but not ibrutinib, suggesting the activation of Bax in ibr-7 treated BxPC-3 and SW1990 cells ( Figure 3B). Generally, activated Bax might demolish the function of mitochondrion; thus, we evaluated mitochondrial membrane potential using JC-1 stain. After treatment with indicated compounds for 16 h, the proportion of red to green cells, which indicated depolarized membrane potential, significantly increased only after exposure to ibr-7 ( Figure 3C). These results suggested that mitochondria were involved in ibr-7-induced apoptosis. In addition, a pan-caspase inhibitor V-ZAD-FMK was pretreated with BxPC-3 and SW1990 cells before incubation with ibr-7.
As a result, an approximate 20% increase in survival cells was seen after V-ZAD-FMK pretreatment.

| Ibr-7 and gemcitabine synergistically inhibited the growth of PDAC cells through repressing mTOR/p79S6K pathway
Gemcitabine has been used as the first-line chemotherapy against pancreatic cancer in the last decades, whereas few agents showed synergistic effects when combined with gemcitabine in clinic trials. Therefore, it is in urgent need to explore novel agents that sensitize the efficacy of gemcitabine in PDAC cells. In our study, the combination treatment of ibr-7 and gemcitabine resulted in 42.1% and 37.4% apoptotic cells in BxPC-3 and SW1990 cells respectively ( Figure 4A). In addition, the occurrence of apoptosis caused by the combination treatment of ibr-7 and gemcitabine was validated by the appearance of apoptotic bodies ( Figure 4B).

| Ibr-7 exerted its synergistic effects with gemcitabine via suppression of TRIM32
To find out key proteins that play decisive roles in the synergistic effect of ibr-7 combined with gemcitabine, cluster of genes that differentially expressed in BxPC-3 cells after gemcitabine or combination treatment were identified by using mRNA sequencing. A total of 506 mRNAs were shown to be significantly changed between combination and gemcitabine group. Using a twofold change and the P and q values at 0.00 as a standard, 325 and 181 mRNAs were shown to be up-regulated and downregulated in combination treatment comparing with gemcitabine group respectively ( Figure 5A-B).
Notably, TRIM32 was the most significantly downregulated gene among these detected genes. Using Cancer Genome Atlas (TCGA, https://www.cancer.gov) database, TRIM32 mRNA was significantly increased in PDAC patients versus normal tissues ( Figure 5C). In addition, the elevated expression of TRIM32 was closely associated with exposure to gemcitabine in a dose-dependent manner, while either ibr-7 or the combination treatment could restore the TRIM32 mRNA level ( Figure 5D). On the contrary, ibrutinib could not suppress TRIM32, but dramatically increase the mRNA expression of TRIM32 ( Figure 5E). Moreover, we aimed to determine the protein expression of TRIM32 after ibr-7 or the combination treatment. As shown in Figure 5F, the protein level of TRIM32 considerably decreased after combination treatment for 24 h. To further understand the transcriptional regulation of TRIM32, cells were pretreated with decitabine (DAC), which was known as a DNA demethylating agent, for 2 h before incubation with ibr-7, gemcitabine or the combination.
As a result, pretreatment with dcecitabine allowed restoration of TRIM32 protein level after exposure to the combination treatment, suggesting that diminished TRIM32 mRNA transcription level was attributed to its DNA methylation ( Figure 5G).

F I G U R E 2 Ibr-7 induced apoptosis in BxPC-3 and SW1990 cells. (A)
Cells were treated with ibr-7 or ibrutinib for 24 h before collection. Then, cells were stained by Annexin V/PI and analysed by flow cytometry. (B) BxPC-3 cells were treated with 2, 4 and 8 µM of ibr-7 and 8 µM of ibrutinib for 24 h before fixation and stained with DAPI stain. Fluorescence was observed by using microscopy (Nikon Eclipse Ti). The magnification is ×400. Scale bar =20 µm. Three independent experiments were performed, and data were presented as mean ± SD. Student's t test was used to make a comparison between two groups. ** p < 0.01, *** p < 0.001

| TRIM32 played a key role in the progression of PDAC cells
To elaborate the role of TRIM32 in malignant characteristics of PDAC, we used RNA interference to diminish the expression of TRIM32 ( Figure 6A). As a result, knocking down of TRIM32 not only impeded the proliferation of BxPC-3 cells, but also suppressed the colony formation of BxPC-3 cells (Figure 6B-C). Next, we intended to understand the influence of TRIM32 silencing on mTOR/p70S6K pathway. In BxPC-3 cells, silencing TRIM32 significantly suppressed the phosphorylated p-p70S6, without affecting total or phosphorylated mTOR. In SW1990 cells, the inhibitory effects on mTOR were also prominent after TRIM32 silencing ( Figure 6D). Moreover, either TRIM32 silencing or the addition of rapamycin could potentiate the inhibition of mTOR/p70S6K pathway after the combination treatment with ibr-7 and gemcitabine. These data suggested a positive feedback loop between TRIM32 and mTOR/p70S6K pathway.
Then, we aimed to investigate the role of TRIM32 in drug sensitivity of PDAC cells. Interestingly, silencing of TRIM32 could enhance the cytotoxicity of gemcitabine in BxPC-3 cells ( Figure 6E), while overexpression of TRIM32 partially attenuated the inhibitory effects of gemcitabine single treatment but not the combination treatment ( Figure 6F). In addition, excessive TRIM32 could enhance the invasiveness of BxPC-3 cells ( Figure 6G) and meanwhile accelerated the proliferation of BxPC-3 cells ( Figure 6H). Interestingly, in three PDAC cells with established sustained overexpression of TRIM32, total or phophorylated mTOR was found to be downregulated compared with those transfected with empty vector ( Figure 6I). Three independent experiments were performed, and data were presented as mean ± SD. Student's t test was used to make a comparison between two groups. ** p < 0.01

| DISCUSS ION
In the era of precision medicine, targeted therapy for pancreatic cancer is facing critical challenges. On one hand, the stroma of PDAC microenvironment provides a natural barrier against most of therapeutic drugs and creates metastatic niche for tumour progression. 21,22 On the other hand, PDAC cells are naturally insensitive to current chemotherapy or targeted drugs. 23,24 Ibr-7 was a derivative of ibrutinib that obtained in our previous work. 17 Preliminary studies showed that ibr-7 could inhibit the proliferation of PANC-1 and Capan2 cells in vitro, and enhanced radiosensitivity in a p-EGFR dependent manner. 18 However, several questions remained to be fully illustrated: the underlying mechanisms of ibr-7's inhibitory activity in PDAC cells; whether ibr-7 was capable to improve the efficacy of gemcitabine; and the potential molecular targets that sensitizing gemcitabine in PDAC cells. Herein, we not only explored the molecular mechanisms of anti-PDAC activity of ibr-7 but also investigated the regulatory loop between TRIM32 and mTOR/p70S6K pathway, thus preliminarily demonstrating the potential role of TRIM32 as a novel molecular target for sensitizing gemcitabine in PDAC cells.
First of all, we examined the anti-proliferation activity of ibrutinib and ibr-7 in four PDAC cell lines in vitro. The IC50 values of ibr-7 ranged from 0.8 to 3.5 μM, which were 10-fold to 60-fold less than that of ibrutinib (Table 1). To explore the underlying Three independent experiments were performed, and data were presented as mean ± SD. Student's t test was used to make a comparison between two groups. n.s.=non-significant, ** p < 0.01 ( Figure 4C-E). By analysing the differential gene expression, TRIM32 was the most downregulated gene in 959 differential genes while comparing combinatorial treatment and gemcitabine single treatment ( Figure 5A-B). Indeed, TRIM32 was significantly downregulated after the combinatorial treatment of gemcitabine and ibr-7, but not ibrutinib ( Figure 5D-E). It was reported that TRIM32 played a pro-apoptotic role via destabilizing mitochondrial membrane potential and degradation of XIAP in normal cells. 32,33 In the context of cancer, TRIM32 was able to promote the proliferation and motility of lung, gastric, squamous cancer cells or contribute to cisplatin resistance in colorectal cancer, whereas the role of TRIM32 in PDAC cells remained unknown. 3 4-38 In our study, we found that the combination treatment of ibr-7 and gemcitabine could diminish TRIM32 at a transcriptional level, probably through enhancing the methylation status of TRIM32 ( Figure 5F,G). 39,40 F I G U R E 6 Role of TRIM32 in malignant properties of PDAC cells. (A) BxPC-3 cells were transfected with small interfering RNA (siRNA) targeting TRIM32 (SiTRIM32) or scramble siRNA (SiControl). After cells were transfected for 24 h, proteins were collected and analysed by Western blotting assay. (B) BxPC-3 cells were seeded in 96-well plates at a density of 1 × 10 3 /well and cultured for 24 h. After transfection with siTRIM32 or siControl, BxPC-3 cell number was counted every day for 9 days. (C) BxPC-3 cells were seeded in 6-well plates at a density of 1 × 10 3 /well and cultured for 24 h. After transfection with siTRIM32 or siControl, the supernatant was removed and cells were cultured for another two weeks. Then, cells were fixed with 4% paraformaldehyde for 15 min and stained with Giemsa solution for 15 min at room temperature. Visible colonies were imaged with a ChemiDoc XPS system. (D) BxPC-3 and SW1990 cells were treated with siTRIM32 or indicated agents for 8 h before Western blotting assay. Gem =gemcitabine, Rapa =rapamycin. (E) BxPC-3 cells were seeded in 96-well plates at a density of 6 × 10 3 /well and cultured for 24 h. Then, cells were transfected with siTRIM32 or siControl for another 24 h. Cells were treated with 8 μM of gemcitabine for 24 h before CCK-8 assay. (F) BxPC-3 cells were seeded in 96-well plates at a density of 6 × 10 3 /well and cultured for 24 h. After transfection with TRIM32, cells were treated with ibr-7 (2 µM), gemcitabine (8 µM, Gem) or the combination for 24 h before CCK-8 assay. (G) BxPC-3 cells were transfected with TRIM32 and incubated with a combination of ibr-7 (2 µM), gemcitabine (8 µM, Gem) or the combination for 24 h before migration assay. (H) BxPC-3 cells were cultured in 24-well plates at a density of 1 × 10 4 / well and transfected with TRIM32 for 24 h. Cells were incubated in culturing medium for 5 days, and cell number was counted every day by using Counterstar (Shanghai, China). (I) BxPC-3, SW1990 and CFPAC-1 cells were transfected with empty vector (EV) or TRIM32 (OE, overexpression) and cultured for 48 h. Then, cells were lysed, and indicated proteins were examined by Western blotting assay. Three independent experiments were performed, and data were presented as mean ± SD. Student's t test was used to make a comparison between two groups. * p < 0.05. n.s.=non-significant difference Based on the aforementioned data, we assumed that the direct inhibition of mTOR/p70S6K pathway caused by ibr-7 probably contributed to the suppression of TRIM32. In another aspects, whether TRIM32 possessed a reciprocal feedback loop with mTOR/p70S6K in PDAC cells remained to be illuminated. It was reported that TRIM32 reduced PI3K-Akt-FoxO signalling by promoting plakoglobin-PI3K dissociation in muscle atrophy, 41 and TRIM32 deficiency was found to cause hypoactive mTOR in autism spectrum disorder mice model. 42 In gastric cancer cells, TRIM32 was able to promote the AKT activity and glucose transportation. 43 In this work, knocking down TRIM32 significantly impeded the proliferation and diminished the colony formation of BxPC-3 cells. Moreover, silencing TRIM32 could significantly suppressed the phosphorylated p-p70S6 or mTOR in BxPC-3 and SW1990 cells, respectively ( Figure 6D), which was in consistent with previous reports. Nevertheless, different situations were found in stable transfected PDAC cells with overexpression of TRIM32. Although overexpression of TRIM32 enhanced the invasion and growth of BxPC-3 cells, a repression of either mTOR or p70S6 was seen in all three PDAC cell lines ( Figure 6I). This phenomenon could be explained by the functional role of TRIM32 as an E3 ligase, the residual of which resulted in increased proteasomal degradation in PDAC cells. 44,45 In summary, our study showed that ibr-7 alone or combined with