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DIAPH3 promotes the tumorigenesis of lung adenocarcinoma
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DIAPH3 promotes the tumorigenesis of lung adenocarcinoma Guo Xianga,b, He Weiweia,b, Gao Erjib, Ma Haitaoa,∗ a b
The First Affiliated Hospital of Soochow University, Suzhou, 215006, China Department of Thoracic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer DIAPH3 MEKK-MEK-ERK STK38
Aberrant activation of MEKK-MEK-ERK signaling is frequently observed in lung cancer. Several inhibitors, which target this pathway, have shown clinical potential for the lung cancer treatment. Better understanding the regulation of this pathway would help the development of treatment strategies. In this study, we have identified the DIAPH3 as an up-regulated gene in lung adenocarcinoma. DIAPH3 promoted the growth of lung cancer cells both in the liquid culture and in the soft agar, and knockdown DIAPH3 inhibited the tumorigenesis both in the nude mice and in the de novo mouse model. In the molecular mechanism study, DIAPH3 was identified as the binding protein of STK38, impaired the interaction between STK38 and MEKK, and activated ERK signaling. Taken together, this study demonstrated the oncogenic roles of DIAPH3 in the tumorigenesis of lung cancer by interacting with STK38.
1. Introduction Hyperactivation of ERK signaling is frequently observed due to the mutation of Kras [1]. Although recent studies have shown that the potent inhibitor ARS-853 could inhibit KrasG12C, targeting other types of Kras mutation in lung cancer has still been challenging [2,3]. Targeting the downstream effectors (CRAF, ERK and MEK) has been considered as the strategy for the lung cancer treatment [4]. Therefore, further understanding the regulation of MEKK-MEK-ERK signaling would help to design new therapeutic strategies. STK38 is a member of the AGC serine/threonine kinase family. The kinase activity of this protein is regulated by autophosphorylation and phosphorylation by other upstream kinases [5,6]. Several studies have shown that STK38 cross-talked with MEKK-MEK-ERK pathways [7]. In the gastric cancer, STK38 has been shown to negatively regulate the stability of MEKK protein and inhibit the MEKK-MEK-ERK signaling, which was antagonized by the Kir2.1 protein [8]. In addition, the regulation of STK38/MEKK interaction was attenuated by the SENP2 protein which mediated the SUMOylation of STK38 [7]. These observations suggested that the interaction between STK38 and MEKK was critical for the cancer cells and should be tightly regulated from different levels. DIAPH3 is a member of the diaphanous subfamily [9]. Members of this family are involved in actin remodeling and the regulation of cell movement and adhesion [10–12]. Several studies have shown the roles of DIAPH3 in the development and disease [13,14]. A transient
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expression of DIAPH3 was found in the developing murine cerebral cortex, indicating it had a function in brain development [15]. Moreover, increased activity of DIAPH3 was correlated with the hearing defects in humans with auditory neuropathy [14,16]. The functions of DIAPH3 in the cell motility seemed to depend on the context. DIAPH3, in prostate and breast cancer cells, induced nuclear shape instability, with a corresponding gain in malignant properties [17]. Consistent with these observations, DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/ TCF signaling [11]. On the other hand, silencing DIAPH3 evoked amoeboid properties, increased invasion and promoted metastasis in mice [12,18], and Diaphanous-related formin-3 overexpression inhibits the migration and invasion of triple-negative breast cancer by inhibiting RhoA-GTP expression [19]. However, the roles of DIAPH3 in the tumorigenesis of lung cancer were poorly understood. In this study, the expression pattern and the functions of DIAPH3 in the lung adenocarcinoma were examined, and the molecular mechanism was studied. 2. Materials and methods 2.1. Mining the GEPIA database The expression and survival analysis of DIAPH3 was performed using online software, Gene Expression Profiling Interactive Analysis (GEPIA). Lung adenocarcinoma (LUAD) and lung squamous cell
Corresponding author. E-mail address: [email protected] (M. Haitao).
https://doi.org/10.1016/j.yexcr.2019.111662 Received 19 June 2019; Received in revised form 30 September 2019; Accepted 1 October 2019 0014-4827/ © 2019 Published by Elsevier Inc.
Please cite this article as: Guo Xiang, et al., Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2019.111662
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2.7. MTT
carcinoma (LUSC) matched the Cancer Genome Atlas (TCGA) normal and Genotype-Tissue Expression (GTEx) data were used for the expression analysis, and log2 (TPM+1) was used for log-scale. Overall survival (OS) analysis was performed based on gene expression, using Log-rank test, a.k.a the Mantel-Cox test, for hypothesis test on GEPIA.
Briefly, 103 cells per wells were seeded in the 96-well plate, and incubated with a total of 200 μl medium. On the next day, cells were incubated with additional 20 μl MTT solution for 4 h. After the supernatant was removed, 200 μl DMSO was added and the OD540 nm was examined.
2.2. Clinical sample The clinical samples were obtained from Shanghai Jiao Tong University Affiliated Sixth People's Hospital with the written consent forms. Samples were stored at −80 °C. The histopathology was evaluated by two pathologists.
2.8. Soft agar The bottom layer contained 0.5% agarose and 10% FBS in DMEM, and was used to coat the 12-well plate. The upper layer in the 12-well plates contained 0.35% agarose and 10% FBS in DMEM. 2˟103 cells were suspended in the upper layer. The colonies were photographed and counted after 14 days’ incubation. All experiments were performed for at least three times.
2.3. Cell culture and transfection The lung cancer cells were obtained from the cell bank in the Chinese Academy of Science. KPC cells were the primary culture of the lungs from P53f/f; KrasG12D mice treated with Ad-Cre virus, and were the gifts from Dr Ji (the Institutes for Biological Science, Chinese Academy of Science, Shanghai, China). Cells were cultured in DMEM with 10% FBS and antibiotics (Penicillin and streptomycin) at a 37 °C atmosphere of 5% CO2. The cells were plated in the 100 mm dish 18 h before transfection. Lipofectamine 2000 was used to deliver the plasmids into the lung cancer cells according to the manufacture's instructions. For the establishment of stable cell lines, the transfected cells were incubated with puromycin for a week, and the expression of exogenous protein was examined using western blot.
2.9. Knocking down DIAPH3 The lentivirus to knockdown the human DIAPH3 and mouse DIAPH3 was purchased from Genechem (Shanghai, China). For human DIAPH3, sh DIAPH3 1#, 5′-aaaatccaagaaaaagttgta-3’; sh DIAPH3 2#, 5′-aagattgaattggttaaagat-3’. For mouse DIAPH3, sh DIAPH3 1#, 5′aattaatgggcaaatccaaga-3’; sh DIAPH3 2#, 5′-aatgatcgttttataagagag-3’. Cells were incubated with the lentivirus (MOI = 1) for 8 h and selected with puromycin for 7 days. The resistant cells were pooled and the DIAPH3 protein levels were examined using western blot. 2.10. Silver staining and mass spectrum
2.4. IHC 293 T cells were transfected with Flag-DIAPH3 and were harvested using the RIPA buffer 48 h after transfection. After centrifugation, the supernatant was incubated with sepharose beads coupled with the antiFlag antibody for 4 h. After extensive wash, the immunoprecipitates were separated with SDS-PAGE. The gel was stained with the silver staining kit according to the manufacture's instruction. There were DIAPH3 specific bands in the anti-Flag antibody pull-down, but they were not found in the control. The differential bands were cut, and analyzed with mass spectrum (Institute of Biophysics, the Chinese Academy of Science, Beijing, China).
Xylene and the gradient ethanol were used to deparaffinize and rehydrate the sections (5 μm), and 0.3% H2O2 solution was used to block endogenous peroxidase activity. Then, the antigens were retrieved using sodium citrate solution (pH 6.0), non-specific binding was blocked using 5% BSA solution. Next, the sections were stained with DIAPH3 antibody and visualized with the secondary antibody (Envision, Gene Technology). Then, the slides were developed with DAB and counterstained with hematoxylin. 2.5. Western blot
2.11. GST pull down Cells were harvested using the RIPA buffer, and the cell lysate was put on the ice for 20 min. Then, the cell lysate was centrifugated for 20 min with 12,000 rpm at 4 °C. The supernatant was collected and the concentration was measured using the Bradford. SDS-PAGE was performed. The PDVF membrane was used for the protein transferred from the SDS-PAGE, blocked with the 5% milk (1 h; room temperature) and incubated with the primary antibodies for at least 8 h. After washing for 2–3 times, the membrane was incubated with the HRP-coupled IgG at room temperature for 1 h. After washing, the signals were detected with the ECL kit.
The fusion protein GST-STK38 was expressed in E. coli, captured by the sepharose 4B beads and added to the cell lysates overnight. The binding protein of GST or GST-STK38 was eluted with loading buffer and examined using western blot. 2.12. Immunoprecipitation The indicated plasmids were transfected into cells using lipofectamine 6000. The transfected cells were harvested using the lysis buffer. After centrifugation, the supernatant was collected and the primary antibody was added. After incubation overnight, the primary antibody was pulled down by protein A beads. Four hours later, the protein A beads were harvested and washed. The binding protein was eluted with the loading buffer and examined using western blot.
2.6. KPC mouse model All mice were housed in a pathogen-free environment in Shanghai Jiaotong University. All experimental protocols were approved by the Institutional Committee for Animal Care and Use at Shanghai Jiaotong University. All animal work was performed in accordance with the approved protocol. The Ad-Cre virus was purchased from Obio (Shanghai, China) and delivered into the mice (P53f/f; KrasG12D) though nostril inhalation (109 per mice). Two-weeks later, the lentivirus which mediated the knockdown of DIAPH3 was purchased from Genechem (Shanghai, China) and delivered into the mice though nostril inhalation (109 per mice). 12 weeks later, the mice were sacrificed and the lungs were examined.
3. Results 3.1. The DIAPH3 expression levels were elevated in lung adenocarcinoma (LUAD) To study the expression pattern of DIAPH3 in the lung cancer, we first turned to the GEPIA database (http://gepia.cancer-pku.cn/), and analyzed the correlation between DIAPH3 expression and the survival 2
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Fig. 1. Elevated DIAPH3 expression levels were observed in lung adenocarcinoma. (A) The correlations between the DIAPH3 expression and the survival of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and the combination of LUAD and LUSC were mined using the GEPIA database. (B) Immunohistochemistry was performed to examine the DIAPH3 protein levels in LUAD tissues and control tissues. Representative images were shown. (C) Statistic analysis of (B). The IHC signal intensity was scored and analyzed. **, P < 0.01. (D) Western blot was performed to examine the DIAPH3 protein levels in normal bronchus cell Bease-2B and lung cancer cells. (E) The DIAPH3 protein level in the P53f/f; KrasG12D mice was assessed after the administration of the Ad-Cre virus.
1) were examined. Higher DIAPH3 protein level was found in lung cancer cells (Fig. 1D). Ad-Cre; P53f/f; KrasG12D mouse model has been used as a tool to study the LUAD. Significantly, administration of the mice model with Ad-Cre induced the expression of DIAPH3, possibly accompanied with the tumorigenesis (Fig. 1E). These observations demonstrated the upregulation of DIAPH3 in the lung adenocarcinoma.
of the LUAD patients and LUSC (lung squamous cell carcinoma) patients. As shown in Fig. 1A, in the cohort of 476 LUAD patients, high DIAPH3 expression inversely correlated with the outcome. However, the correlation between DIAPH3 expression and the survival of 481 LUSC patients was of no statistical significance. We next examined the protein levels of DIAPH3 in 30 LUAD tissues as well as the paired noncancerous tissues using immunohistochemistry (IHC). Weak staining of DIAPH3 was found in the normal lung tissues, and higher DIAPH3 protein levels were observed in LUAD tissues (Fig. 1B–C). In addition, the DIAPH3 protein levels in normal bronchial cells (Bease-2B) and a panel of lung cancer cells (A549, H520, H23, H460, H1299 and SPC-A-
3.2. DIAPH3 promoted the growth and colony formation of LUAD cells The functions of DIAPH3 in the lung cancer cells were evaluated 3
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Fig. 2. DIAPH3 promoted the growth of LUAD cells. (A) The A549, SPC-A-1 and KPC cells were transfected with Flag-DIAPH3 plasmids and the expression of exogenous were examined using the anti-Flag antibody. (B) MTT assay was performed to evaluate the effects of DIAPH3 on the growth of LUAD cells in the liquid culture. (C) Soft agar assay was performed to evaluate the effects of DIAPH3 on the growth of LUAD cells in the soft agar. (D) Statistic analysis of (C). **, P < 0.01.
using the gain-of-function assay and loss-of-function assay. The flagtagged DIAPH3 (Flag-DIAPH3) was ectopically expressed in A549, SPCA-1 and the primary culture KPC cells (Fig. 2A). The functions of DIAPH3 in the growth of lung cancer cells were examined using MTT assay and soft agar assay. As shown in Fig. 2B, overexpression of DIAPH3 significantly promoted the growth of A549, SPC-A-1 and KPC cells in the liquid culture (Fig. 2B). Moreover, overexpression of DIAPH3 boosted the anchorage-independent growth of A549, SPC-A-1 and KPC cells (Fig. 2C–D).
To further confirm the functions of DIAPH3 in the lung cancer, we knocked down the expression of DIAPH3 in A549, SPC-A-1, KPC and NCI-H358 cells (Fig. 3A). Silencing the expression of DIAPH3 in A549, SPC-A-1, KPC and NCI-H358 cells inhibited the growth of lung cancer cells evaluated by MTT assay (Fig. 3B) as well as the anchorage-independent growth (Fig. 3C–D). To rule out the off-target effects, the sh RNA-resistant DIAPH3 (rDIAPH3) was used to rescue the phenotype. Obviously, rDIAPH3 effectively rescued the inhibition of cell growth in the liquid culture and soft agar induced by DIAPH3 knockdown 4
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Fig. 3. Knockdown of DIAPH3 hindered the growth of LUAD cells. (A) The A549, SPC-A-1, KPC and NCI-H358 cells were knocked down the expression of DIAPH3 by two independent shRNA sequences. (B) MTT assay was performed to evaluate the effects of DIAPH3 knockdown on the growth of LUAD cells in the liquid culture. (C) Soft agar assay was performed to evaluate the effects of DIAPH3 knockdown on the growth of LUAD cells in the soft agar. (D) Statistic analysis of (C). **, P < 0.01. (E) The RNAi-resistant DIAPH3 rescued the cell growth defects in the MTT assay. **, P < 0.01. (F–G) The RNAi-resistant DIAPH3 rescued the anchorage-independent cell growth defects in the soft agar assay. **, P < 0.01.
3.3. Knocking down the expression of DIAPH3 inhibited the de novo tumorigenesis
(Fig. 3E–G). In addition, the expression of DIAPH3 in H157 and H520 (lung squamous cell carcinoma cell lines) cells were down-regulated by sh RNA (Fig. S1A). Knocking down of DIAPH3 exerted few effects on the anchorage-independent growth of H157 and H520 cells (Figs. S1B–C). Also, knocking down of DIAPH3 did not affect cell motility in the wound healing assay (Fig. S1D).
The in vitro functions of DIAPH3 prompted us to investigate its roles in vivo. Knocking down the expression of DIAPH3 impaired the tumorigenicity of SPC-A-1 cells in the nude mice, which was demonstrated by the slower tumor growth (Fig. 4A), smaller tumor volume (Fig. 4B) and lighter tumor weight (Fig. 4C). Consistently, overexpression of DIAPH3 in A549 cells promoted the tumorigenesis, which was demonstrated by the tumor weight (Fig. 4D–E).
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Fig. 4. Knockdown of DIAPH3 inhibited the tumorigenesis. (A) The effects of DIAPH3 knockdown on the tumorigenicity of SPC-A-1 cells were evaluated by the tumor growth curve. **, P < 0.01. (B) The image of the tumors shown in (A). (C) The weight of the tumors shown in (A). (D) The effects of DIAPH3 overexpression on the tumorigenicity of A549 cells were evaluated. The image of the tumors was shown. (E) The weight of the tumors shown in (D). (F) The de novo tumorigenesis of lung cancer. The mice were treated with Ad-Cre virus. Two weeks later, the mice were treated with sh DIAPH3 virus. The HE staining was performed 12 weeks after the mice were treated with sh DIAPH3 virus. (G) Statistical analysis of the foci shown in (F).
3.5. STK38 inhibited the growth of LUAD cells
To test the roles of DIAPH3 in the de novo tumorigenesis of lung cancer, the 6-weeks old P53f/f; KrasG12D mice model was treated with the adenovirus which contained the Cre coding sequence (Ad-Cre). Two weeks later, the mice were further treated with sh mDIAPH3 lentivirus. It was found that down-regulation of DIAPH3 impaired the de novo tumorigenesis (Fig. 4F–G).
The functions of STK38 in the tumorigenesis of lung cancer have not been addressed. Mining the GEPIA database showed that the little difference of STK38 expression level was found in the LUAD tissues or LUSC tissues when compared with their counterparts, respectively (Fig. 6A). Also, the expression of STK38 did not correlated with the survival (Fig. 6B). However, overexpression of STK38 in SPC-A-1 and A549 cells inhibited cell growth in the liquid culture and soft agar (Fig. 6C–D). In addition, knockdown of STK38 up-regulated the MEKK protein level (Fig. 6E).
3.4. DIAPH3 interacted with STK38 To understand the molecular mechanisms through which DIAPH3 promoted the lung tumorigenesis, the binding proteins of DIAPH3 were screened using the mass spectrum (Fig. 5A). STK38 was one of the potential candidates. Therefore, we next tested the interaction between DIAPH3 and STK38 using GST pull-down assay and immunoprecipitation assay. As shown in Fig. 5B, the fusion protein GST-STK38 formed a complex with DIAPH3. Also, when the Flag tagged DIAPH3 (FlagDIAPH3) and the HA tagged STK38 (HA-STK38) were overexpressed in SPC-A-1 cells, these proteins were in the same complex (Fig. 5C). Most importantly, the endogenous STK38 was found to form a complex with DIAPH3 in the immunoprecipitation assay (Fig. 5D).
3.6. DIAPH3 rescued the inhibitory effects of STK38 To examine the functional link between DIAPH3 and STK38, we simultaneously over-expressed Flag-DIAPH3 and HA-STK38 in SPC-A1 cells (Fig. 7A). Overexpression of DIAPH3 effectively abolished the inhibitory effects of STK38 on the anchorage-independent growth of SPC-A-1 cells (Fig. 7B). Consistently, Overexpression of DIAPH3 effectively rescued the MEK kinase protein level (Fig. 7C). To understand the molecular mechanism, the interaction between STK38 and MEK kinase 6
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Fig. 5. DIAPH3 interacted with STK38. (A) Silver staining and mass spectrum were performed to screen the binding protein of DIAPH3. 293 T cells were transfected with Flag-DIAPH3 plasmids. 48 h later, the immunoprecipitation was performed using the anti-Flag antibody. The immunoprecipitates were separated with 10% SDSPAGE, and the differential band at about 60 KD (arrow) was cut for mass spectrum analysis. (B) GST pull-down assay was performed to examine the interaction between DIAPH3 and the fusion protein GST-STK38 in SPC-A-1 cells. (C) Immunoprecipitation assay was performed to examine the interaction between DIAPH3 and STK38. Flag-DIAPH3 and HA-STK38 plasmids were transfected into SPC-A-1 cells. 48 h later, the immunoprecipitation was performed using the anti-HA antibody. (D) Immunoprecipitation assay was performed to examine the interaction between endogenous DIAPH3 and STK38 in SPC-A-1 cells.
4. Discussion
was evaluated. Interestingly, DIAPH3 disrupted the interaction between STK38 and MEK kinase (Fig. 7D). Moreover, the rescue effects of DIAPH3 on the inhibitory roles of STK38 could be attenuated by the ERK inhibitor, LY3214996 (Fig. 7B), suggesting that DIAPH3 rescued the inhibitory effects of STK38 and activated ERK signaling.
Aberrant activation of MEK-ERK signaling is very common in the lung cancer [20]. However, how the MEK-ERK signaling is regulated does not fully understood. In this study, we have found that DIAPH3 upregulated the MEKK protein level by inhibiting the interaction between
Fig. 6. STK38 inhibited the growth of LUAD cells. (A) Mining the GEPIA database revealed that no difference of STK38 expression between the LUAD tissues and adjacent normal tissues. (B) Mining the GEPIA database revealed that no correlation between STK38 expression and the survival of patients. (C) STK38 hindered the growth of SPC-A-1 in the MTT assay. (D) STK38 hindered the anchorage-independent growth of A549 and SPC-A-1 cells in the soft agar assay. (E) STK38 knockdown up-regulated the MEKK protein level. **, P < 0.01. 7
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Fig. 7. DIAPH3 attenuated the interaction between STK38 and MEKK. (A) Simultaneously expression of Flag-DIAPH3 and HA-STK38 in SPC-A-1 cells was examined using western blot. (B) The rescue effects of DIAPH3 on the anchorage-independent growth of SPC-A-1 cells were examined using soft agar. The ERK inhibitor, LY3214996, was used to examine the roles of ERK signaling. (C) The rescue effects of DIAPH3 on the expression of MEKK were examined using western blot. (D) DIAPH3 disrupted the interaction between MEKK and STK38. **, P < 0.01.
the up-regulation of DIAPH3, the interaction between the STK38-MEKK was attenuated, thus the MEK/ERK signaling was activated. In summary, we have demonstrated the oncogenic roles of DIAPH3 in lung cancer by interacting with STK38 in this study, and suggested that DIAPH3 might be a therapeutic target.
STK38 and MEK, thus promoted the tumorigenicity of lung cancer cells. These results clearly demonstrated the oncogenic roles of DIAPH3 in the lung cancer. Several observations were provocative for a few reasons. First, the up-regulation of DIAPH3 was only observed in the lung adenocarcinoma. One of the good explanations for this might be that DIAPH3 expression was negatively regulated by P53. This hypothesis was based on two aspects: 1. The Ad-Cre; P53f/f; KrasG12D lung adenocarcinoma mice showed higher DIAPH3 protein levels; 2. The loss-of-function mutation of P53 is very common in lung adenocarcinoma than the lung squamous cell carcinoma. Based on these observations, DIAPH3 might be a bio-marker for lung adenocarcinoma. Further, DIAPH3 promoted the anchorage-independent growth of lung cancer cells. It has been reported that DIAPH3 regulated the dynamics of cytoskeleton and cell motility. Although we did not examine the functions of DIAPH3 in the migration and metastasis of lung cancer cells, it could not rule out the possibility that DIAPH3 regulated the migration and metastasis of lung cancer cells. One of the most interesting findings of this study was the attenuated interaction between STK38 and MEKK by DIAPH3. It has been known that STK38 interacted with MEKK and inhibited the activation of MEK/ ERK signaling [7]. In the normal cells, the interaction between STK38DIAPH3 and STK38-MEKK reached the balance. In cancer cells, due to
Ethics approval and consent to participate All experimental protocols were approved by Shanghai Jiaotong University institutional committee. Informed consent was obtained from all subjects. The study was reviewed and approved by the China national institutional animal care and use committee.
Consent for publication All authors agreed on the manuscript.
Declaration of competing interest The authors declare that they have no competing interests. There is no conflict of interest. 8
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Appendix A. Supplementary data [11]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yexcr.2019.111662.
[12]
References [13]
[1] M. Cully, Cancer: closing the door on KRAS-mutant lung cancer, Nature reviews, Drug Discov. 15 (2016) 747. [2] R. Hansen, U. Peters, A. Babbar, Y. Chen, J. Feng, M.R. Janes, L.S. Li, P. Ren, Y. Liu, P.P. Zarrinkar, The reactivity-driven biochemical mechanism of covalent KRAS (G12C) inhibitors, Nat. Struct. Mol. Biol. 25 (2018) 454–462. [3] M.P. Patricelli, M.R. Janes, L.S. Li, R. Hansen, U. Peters, L.V. Kessler, Y. Chen, J.M. Kucharski, J. Feng, T. Ely, J.H. Chen, S.J. Firdaus, A. Babbar, P. Ren, Y. Liu, Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state, Cancer Discov. 6 (2016) 316–329. [4] K. Nawaz, R.M. Webster, The non-small-cell lung cancer drug market, Nature reviews, Drug Discov. 15 (2016) 229–230. [5] C. Joffre, P. Codogno, M. Fanto, A. Hergovich, J. Camonis, STK38 at the crossroad between autophagy and apoptosis, Autophagy 12 (2016) 594–595. [6] D.D. Shi, H. Shi, D. Lu, R. Li, Y. Zhang, J. Zhang, NDR1/STK38 potentiates NFkappaB activation by its kinase activity, Cell Biochem. Funct. 30 (2012) 664–670. [7] N. Xiao, H. Li, W. Yu, C. Gu, H. Fang, Y. Peng, H. Mao, Y. Fang, W. Ni, M. Yao, SUMO-specific protease 2 (SENP2) suppresses keratinocyte migration by targeting NDR1 for de-SUMOylation, FASEB J. : Off. Publ. Fed. Am. Soc. Exp. Biol. 33 (2019) 163–174. [8] C.D. Ji, Y.X. Wang, D.F. Xiang, Q. Liu, Z.H. Zhou, F. Qian, L. Yang, Y. Ren, W. Cui, S.L. Xu, X.L. Zhao, X. Zhang, Y. Wang, P. Zhang, J.M. Wang, Y.H. Cui, X.W. Bian, Kir2.1 interaction with Stk38 promotes invasion and metastasis of human gastric cancer by enhancing MEKK2-MEK1/2-ERK1/2 signaling, Cancer Res. 78 (2018) 3041–3053. [9] M. Katoh, M. Katoh, Identification and characterization of human DIAPH3 gene in silico, Int. J. Mol. Med. 13 (2004) 473–478. [10] F. Calvo, N. Ege, A. Grande-Garcia, S. Hooper, R.P. Jenkins, S.I. Chaudhry, K. Harrington, P. Williamson, E. Moeendarbary, G. Charras, E. Sahai, Mechanotransduction and YAP-dependent matrix remodelling is required for the
[14]
[15]
[16]
[17]
[18]
[19]
[20]
9
generation and maintenance of cancer-associated fibroblasts, Nat. Cell Biol. 15 (2013) 637–646. L. Dong, Z. Li, L. Xue, G. Li, C. Zhang, Z. Cai, H. Li, R. Guo, DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling, Mol. Cell. Biochem. 438 (2018) 183–190. M.H. Hager, S. Morley, D.R. Bielenberg, S. Gao, M. Morello, I.N. Holcomb, W. Liu, G. Mouneimne, F. Demichelis, J. Kim, K.R. Solomon, R.M. Adam, W.B. Isaacs, H.N. Higgs, R.L. Vessella, D. Di Vizio, M.R. Freeman, DIAPH3 governs the cellular transition to the amoeboid tumour phenotype, EMBO Mol. Med. 4 (2012) 743–760. S.H. Bae, J.I. Baek, J.D. Lee, M.H. Song, T.J. Kwon, S.K. Oh, J.Y. Jeong, J.Y. Choi, K.Y. Lee, U.K. Kim, Genetic analysis of auditory neuropathy spectrum disorder in the Korean population, Gene 522 (2013) 65–69. G.M. Carvalho, P.Z. Ramos, A.M. Castilho, A.C. Guimaraes, E.L. Sartorato, Molecular study of patients with auditory neuropathy, Mol. Med. Rep. 14 (2016) 481–490. J.A. Vorstman, E. van Daalen, G.R. Jalali, E.R. Schmidt, R.J. Pasterkamp, M. de Jonge, E.A. Hennekam, E. Janson, W.G. Staal, B. van der Zwaag, J.P. Burbach, R.S. Kahn, B.S. Emanuel, H. van Engeland, R.A. Ophoff, A double hit implicates DIAPH3 as an autism risk gene, Mol. Psychiatry 16 (2011) 442–451. R. Lang-Roth, E. Fischer-Krall, C. Kornblum, G. Nurnberg, D. Meschede, I. Goebel, P. Nurnberg, D. Beutner, C. Kubisch, M. Walger, A.E. Volk, AUNA2: a novel type of non-syndromic slowly progressive auditory synaptopathy/auditory neuropathy with autosomal-dominant inheritance, Audiol. Neuro. Otol. 22 (2017) 30–40. M. Reis-Sobreiro, J.F. Chen, T. Novitskaya, S. You, S. Morley, K. Steadman, N.K. Gill, A. Eskaros, M. Rotinen, C.Y. Chu, L.W.K. Chung, H. Tanaka, W. Yang, B.S. Knudsen, H.R. Tseng, A.C. Rowat, E.M. Posadas, A. Zijlstra, D. Di Vizio, M.R. Freeman, Emerin deregulation links nuclear shape instability to metastatic potential, Cancer Res. 78 (2018) 6086–6097. J. Stastna, X. Pan, H. Wang, A. Kollmannsperger, S. Kutscheidt, V. Lohmann, R. Grosse, O.T. Fackler, Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation, Cell Res. 22 (2012) 728–745. J. Jiang, Diaphanous-related formin-3 overexpression inhibits the migration and invasion of triple-negative breast cancer by inhibiting RhoA-GTP expression, Biomed. pharmacother. 94 (2017) 439–445. C. Ferte, F. Andre, J.C. Soria, Molecular circuits of solid tumors: prognostic and predictive tools for bedside use, Nature reviews, Clin. Oncol. 7 (2010) 367–380.
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Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr
DIAPH3 promotes the tumorigenesis of lung adenocarcinoma Guo Xianga,b, He Weiweia,b, Gao Erjib, Ma Haitaoa,∗ a b
The First Affiliated Hospital of Soochow University, Suzhou, 215006, China Department of Thoracic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer DIAPH3 MEKK-MEK-ERK STK38
Aberrant activation of MEKK-MEK-ERK signaling is frequently observed in lung cancer. Several inhibitors, which target this pathway, have shown clinical potential for the lung cancer treatment. Better understanding the regulation of this pathway would help the development of treatment strategies. In this study, we have identified the DIAPH3 as an up-regulated gene in lung adenocarcinoma. DIAPH3 promoted the growth of lung cancer cells both in the liquid culture and in the soft agar, and knockdown DIAPH3 inhibited the tumorigenesis both in the nude mice and in the de novo mouse model. In the molecular mechanism study, DIAPH3 was identified as the binding protein of STK38, impaired the interaction between STK38 and MEKK, and activated ERK signaling. Taken together, this study demonstrated the oncogenic roles of DIAPH3 in the tumorigenesis of lung cancer by interacting with STK38.
1. Introduction Hyperactivation of ERK signaling is frequently observed due to the mutation of Kras [1]. Although recent studies have shown that the potent inhibitor ARS-853 could inhibit KrasG12C, targeting other types of Kras mutation in lung cancer has still been challenging [2,3]. Targeting the downstream effectors (CRAF, ERK and MEK) has been considered as the strategy for the lung cancer treatment [4]. Therefore, further understanding the regulation of MEKK-MEK-ERK signaling would help to design new therapeutic strategies. STK38 is a member of the AGC serine/threonine kinase family. The kinase activity of this protein is regulated by autophosphorylation and phosphorylation by other upstream kinases [5,6]. Several studies have shown that STK38 cross-talked with MEKK-MEK-ERK pathways [7]. In the gastric cancer, STK38 has been shown to negatively regulate the stability of MEKK protein and inhibit the MEKK-MEK-ERK signaling, which was antagonized by the Kir2.1 protein [8]. In addition, the regulation of STK38/MEKK interaction was attenuated by the SENP2 protein which mediated the SUMOylation of STK38 [7]. These observations suggested that the interaction between STK38 and MEKK was critical for the cancer cells and should be tightly regulated from different levels. DIAPH3 is a member of the diaphanous subfamily [9]. Members of this family are involved in actin remodeling and the regulation of cell movement and adhesion [10–12]. Several studies have shown the roles of DIAPH3 in the development and disease [13,14]. A transient
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expression of DIAPH3 was found in the developing murine cerebral cortex, indicating it had a function in brain development [15]. Moreover, increased activity of DIAPH3 was correlated with the hearing defects in humans with auditory neuropathy [14,16]. The functions of DIAPH3 in the cell motility seemed to depend on the context. DIAPH3, in prostate and breast cancer cells, induced nuclear shape instability, with a corresponding gain in malignant properties [17]. Consistent with these observations, DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/ TCF signaling [11]. On the other hand, silencing DIAPH3 evoked amoeboid properties, increased invasion and promoted metastasis in mice [12,18], and Diaphanous-related formin-3 overexpression inhibits the migration and invasion of triple-negative breast cancer by inhibiting RhoA-GTP expression [19]. However, the roles of DIAPH3 in the tumorigenesis of lung cancer were poorly understood. In this study, the expression pattern and the functions of DIAPH3 in the lung adenocarcinoma were examined, and the molecular mechanism was studied. 2. Materials and methods 2.1. Mining the GEPIA database The expression and survival analysis of DIAPH3 was performed using online software, Gene Expression Profiling Interactive Analysis (GEPIA). Lung adenocarcinoma (LUAD) and lung squamous cell
Corresponding author. E-mail address: [email protected] (M. Haitao).
https://doi.org/10.1016/j.yexcr.2019.111662 Received 19 June 2019; Received in revised form 30 September 2019; Accepted 1 October 2019 0014-4827/ © 2019 Published by Elsevier Inc.
Please cite this article as: Guo Xiang, et al., Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2019.111662
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2.7. MTT
carcinoma (LUSC) matched the Cancer Genome Atlas (TCGA) normal and Genotype-Tissue Expression (GTEx) data were used for the expression analysis, and log2 (TPM+1) was used for log-scale. Overall survival (OS) analysis was performed based on gene expression, using Log-rank test, a.k.a the Mantel-Cox test, for hypothesis test on GEPIA.
Briefly, 103 cells per wells were seeded in the 96-well plate, and incubated with a total of 200 μl medium. On the next day, cells were incubated with additional 20 μl MTT solution for 4 h. After the supernatant was removed, 200 μl DMSO was added and the OD540 nm was examined.
2.2. Clinical sample The clinical samples were obtained from Shanghai Jiao Tong University Affiliated Sixth People's Hospital with the written consent forms. Samples were stored at −80 °C. The histopathology was evaluated by two pathologists.
2.8. Soft agar The bottom layer contained 0.5% agarose and 10% FBS in DMEM, and was used to coat the 12-well plate. The upper layer in the 12-well plates contained 0.35% agarose and 10% FBS in DMEM. 2˟103 cells were suspended in the upper layer. The colonies were photographed and counted after 14 days’ incubation. All experiments were performed for at least three times.
2.3. Cell culture and transfection The lung cancer cells were obtained from the cell bank in the Chinese Academy of Science. KPC cells were the primary culture of the lungs from P53f/f; KrasG12D mice treated with Ad-Cre virus, and were the gifts from Dr Ji (the Institutes for Biological Science, Chinese Academy of Science, Shanghai, China). Cells were cultured in DMEM with 10% FBS and antibiotics (Penicillin and streptomycin) at a 37 °C atmosphere of 5% CO2. The cells were plated in the 100 mm dish 18 h before transfection. Lipofectamine 2000 was used to deliver the plasmids into the lung cancer cells according to the manufacture's instructions. For the establishment of stable cell lines, the transfected cells were incubated with puromycin for a week, and the expression of exogenous protein was examined using western blot.
2.9. Knocking down DIAPH3 The lentivirus to knockdown the human DIAPH3 and mouse DIAPH3 was purchased from Genechem (Shanghai, China). For human DIAPH3, sh DIAPH3 1#, 5′-aaaatccaagaaaaagttgta-3’; sh DIAPH3 2#, 5′-aagattgaattggttaaagat-3’. For mouse DIAPH3, sh DIAPH3 1#, 5′aattaatgggcaaatccaaga-3’; sh DIAPH3 2#, 5′-aatgatcgttttataagagag-3’. Cells were incubated with the lentivirus (MOI = 1) for 8 h and selected with puromycin for 7 days. The resistant cells were pooled and the DIAPH3 protein levels were examined using western blot. 2.10. Silver staining and mass spectrum
2.4. IHC 293 T cells were transfected with Flag-DIAPH3 and were harvested using the RIPA buffer 48 h after transfection. After centrifugation, the supernatant was incubated with sepharose beads coupled with the antiFlag antibody for 4 h. After extensive wash, the immunoprecipitates were separated with SDS-PAGE. The gel was stained with the silver staining kit according to the manufacture's instruction. There were DIAPH3 specific bands in the anti-Flag antibody pull-down, but they were not found in the control. The differential bands were cut, and analyzed with mass spectrum (Institute of Biophysics, the Chinese Academy of Science, Beijing, China).
Xylene and the gradient ethanol were used to deparaffinize and rehydrate the sections (5 μm), and 0.3% H2O2 solution was used to block endogenous peroxidase activity. Then, the antigens were retrieved using sodium citrate solution (pH 6.0), non-specific binding was blocked using 5% BSA solution. Next, the sections were stained with DIAPH3 antibody and visualized with the secondary antibody (Envision, Gene Technology). Then, the slides were developed with DAB and counterstained with hematoxylin. 2.5. Western blot
2.11. GST pull down Cells were harvested using the RIPA buffer, and the cell lysate was put on the ice for 20 min. Then, the cell lysate was centrifugated for 20 min with 12,000 rpm at 4 °C. The supernatant was collected and the concentration was measured using the Bradford. SDS-PAGE was performed. The PDVF membrane was used for the protein transferred from the SDS-PAGE, blocked with the 5% milk (1 h; room temperature) and incubated with the primary antibodies for at least 8 h. After washing for 2–3 times, the membrane was incubated with the HRP-coupled IgG at room temperature for 1 h. After washing, the signals were detected with the ECL kit.
The fusion protein GST-STK38 was expressed in E. coli, captured by the sepharose 4B beads and added to the cell lysates overnight. The binding protein of GST or GST-STK38 was eluted with loading buffer and examined using western blot. 2.12. Immunoprecipitation The indicated plasmids were transfected into cells using lipofectamine 6000. The transfected cells were harvested using the lysis buffer. After centrifugation, the supernatant was collected and the primary antibody was added. After incubation overnight, the primary antibody was pulled down by protein A beads. Four hours later, the protein A beads were harvested and washed. The binding protein was eluted with the loading buffer and examined using western blot.
2.6. KPC mouse model All mice were housed in a pathogen-free environment in Shanghai Jiaotong University. All experimental protocols were approved by the Institutional Committee for Animal Care and Use at Shanghai Jiaotong University. All animal work was performed in accordance with the approved protocol. The Ad-Cre virus was purchased from Obio (Shanghai, China) and delivered into the mice (P53f/f; KrasG12D) though nostril inhalation (109 per mice). Two-weeks later, the lentivirus which mediated the knockdown of DIAPH3 was purchased from Genechem (Shanghai, China) and delivered into the mice though nostril inhalation (109 per mice). 12 weeks later, the mice were sacrificed and the lungs were examined.
3. Results 3.1. The DIAPH3 expression levels were elevated in lung adenocarcinoma (LUAD) To study the expression pattern of DIAPH3 in the lung cancer, we first turned to the GEPIA database (http://gepia.cancer-pku.cn/), and analyzed the correlation between DIAPH3 expression and the survival 2
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Fig. 1. Elevated DIAPH3 expression levels were observed in lung adenocarcinoma. (A) The correlations between the DIAPH3 expression and the survival of lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and the combination of LUAD and LUSC were mined using the GEPIA database. (B) Immunohistochemistry was performed to examine the DIAPH3 protein levels in LUAD tissues and control tissues. Representative images were shown. (C) Statistic analysis of (B). The IHC signal intensity was scored and analyzed. **, P < 0.01. (D) Western blot was performed to examine the DIAPH3 protein levels in normal bronchus cell Bease-2B and lung cancer cells. (E) The DIAPH3 protein level in the P53f/f; KrasG12D mice was assessed after the administration of the Ad-Cre virus.
1) were examined. Higher DIAPH3 protein level was found in lung cancer cells (Fig. 1D). Ad-Cre; P53f/f; KrasG12D mouse model has been used as a tool to study the LUAD. Significantly, administration of the mice model with Ad-Cre induced the expression of DIAPH3, possibly accompanied with the tumorigenesis (Fig. 1E). These observations demonstrated the upregulation of DIAPH3 in the lung adenocarcinoma.
of the LUAD patients and LUSC (lung squamous cell carcinoma) patients. As shown in Fig. 1A, in the cohort of 476 LUAD patients, high DIAPH3 expression inversely correlated with the outcome. However, the correlation between DIAPH3 expression and the survival of 481 LUSC patients was of no statistical significance. We next examined the protein levels of DIAPH3 in 30 LUAD tissues as well as the paired noncancerous tissues using immunohistochemistry (IHC). Weak staining of DIAPH3 was found in the normal lung tissues, and higher DIAPH3 protein levels were observed in LUAD tissues (Fig. 1B–C). In addition, the DIAPH3 protein levels in normal bronchial cells (Bease-2B) and a panel of lung cancer cells (A549, H520, H23, H460, H1299 and SPC-A-
3.2. DIAPH3 promoted the growth and colony formation of LUAD cells The functions of DIAPH3 in the lung cancer cells were evaluated 3
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Fig. 2. DIAPH3 promoted the growth of LUAD cells. (A) The A549, SPC-A-1 and KPC cells were transfected with Flag-DIAPH3 plasmids and the expression of exogenous were examined using the anti-Flag antibody. (B) MTT assay was performed to evaluate the effects of DIAPH3 on the growth of LUAD cells in the liquid culture. (C) Soft agar assay was performed to evaluate the effects of DIAPH3 on the growth of LUAD cells in the soft agar. (D) Statistic analysis of (C). **, P < 0.01.
using the gain-of-function assay and loss-of-function assay. The flagtagged DIAPH3 (Flag-DIAPH3) was ectopically expressed in A549, SPCA-1 and the primary culture KPC cells (Fig. 2A). The functions of DIAPH3 in the growth of lung cancer cells were examined using MTT assay and soft agar assay. As shown in Fig. 2B, overexpression of DIAPH3 significantly promoted the growth of A549, SPC-A-1 and KPC cells in the liquid culture (Fig. 2B). Moreover, overexpression of DIAPH3 boosted the anchorage-independent growth of A549, SPC-A-1 and KPC cells (Fig. 2C–D).
To further confirm the functions of DIAPH3 in the lung cancer, we knocked down the expression of DIAPH3 in A549, SPC-A-1, KPC and NCI-H358 cells (Fig. 3A). Silencing the expression of DIAPH3 in A549, SPC-A-1, KPC and NCI-H358 cells inhibited the growth of lung cancer cells evaluated by MTT assay (Fig. 3B) as well as the anchorage-independent growth (Fig. 3C–D). To rule out the off-target effects, the sh RNA-resistant DIAPH3 (rDIAPH3) was used to rescue the phenotype. Obviously, rDIAPH3 effectively rescued the inhibition of cell growth in the liquid culture and soft agar induced by DIAPH3 knockdown 4
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Fig. 3. Knockdown of DIAPH3 hindered the growth of LUAD cells. (A) The A549, SPC-A-1, KPC and NCI-H358 cells were knocked down the expression of DIAPH3 by two independent shRNA sequences. (B) MTT assay was performed to evaluate the effects of DIAPH3 knockdown on the growth of LUAD cells in the liquid culture. (C) Soft agar assay was performed to evaluate the effects of DIAPH3 knockdown on the growth of LUAD cells in the soft agar. (D) Statistic analysis of (C). **, P < 0.01. (E) The RNAi-resistant DIAPH3 rescued the cell growth defects in the MTT assay. **, P < 0.01. (F–G) The RNAi-resistant DIAPH3 rescued the anchorage-independent cell growth defects in the soft agar assay. **, P < 0.01.
3.3. Knocking down the expression of DIAPH3 inhibited the de novo tumorigenesis
(Fig. 3E–G). In addition, the expression of DIAPH3 in H157 and H520 (lung squamous cell carcinoma cell lines) cells were down-regulated by sh RNA (Fig. S1A). Knocking down of DIAPH3 exerted few effects on the anchorage-independent growth of H157 and H520 cells (Figs. S1B–C). Also, knocking down of DIAPH3 did not affect cell motility in the wound healing assay (Fig. S1D).
The in vitro functions of DIAPH3 prompted us to investigate its roles in vivo. Knocking down the expression of DIAPH3 impaired the tumorigenicity of SPC-A-1 cells in the nude mice, which was demonstrated by the slower tumor growth (Fig. 4A), smaller tumor volume (Fig. 4B) and lighter tumor weight (Fig. 4C). Consistently, overexpression of DIAPH3 in A549 cells promoted the tumorigenesis, which was demonstrated by the tumor weight (Fig. 4D–E).
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Fig. 4. Knockdown of DIAPH3 inhibited the tumorigenesis. (A) The effects of DIAPH3 knockdown on the tumorigenicity of SPC-A-1 cells were evaluated by the tumor growth curve. **, P < 0.01. (B) The image of the tumors shown in (A). (C) The weight of the tumors shown in (A). (D) The effects of DIAPH3 overexpression on the tumorigenicity of A549 cells were evaluated. The image of the tumors was shown. (E) The weight of the tumors shown in (D). (F) The de novo tumorigenesis of lung cancer. The mice were treated with Ad-Cre virus. Two weeks later, the mice were treated with sh DIAPH3 virus. The HE staining was performed 12 weeks after the mice were treated with sh DIAPH3 virus. (G) Statistical analysis of the foci shown in (F).
3.5. STK38 inhibited the growth of LUAD cells
To test the roles of DIAPH3 in the de novo tumorigenesis of lung cancer, the 6-weeks old P53f/f; KrasG12D mice model was treated with the adenovirus which contained the Cre coding sequence (Ad-Cre). Two weeks later, the mice were further treated with sh mDIAPH3 lentivirus. It was found that down-regulation of DIAPH3 impaired the de novo tumorigenesis (Fig. 4F–G).
The functions of STK38 in the tumorigenesis of lung cancer have not been addressed. Mining the GEPIA database showed that the little difference of STK38 expression level was found in the LUAD tissues or LUSC tissues when compared with their counterparts, respectively (Fig. 6A). Also, the expression of STK38 did not correlated with the survival (Fig. 6B). However, overexpression of STK38 in SPC-A-1 and A549 cells inhibited cell growth in the liquid culture and soft agar (Fig. 6C–D). In addition, knockdown of STK38 up-regulated the MEKK protein level (Fig. 6E).
3.4. DIAPH3 interacted with STK38 To understand the molecular mechanisms through which DIAPH3 promoted the lung tumorigenesis, the binding proteins of DIAPH3 were screened using the mass spectrum (Fig. 5A). STK38 was one of the potential candidates. Therefore, we next tested the interaction between DIAPH3 and STK38 using GST pull-down assay and immunoprecipitation assay. As shown in Fig. 5B, the fusion protein GST-STK38 formed a complex with DIAPH3. Also, when the Flag tagged DIAPH3 (FlagDIAPH3) and the HA tagged STK38 (HA-STK38) were overexpressed in SPC-A-1 cells, these proteins were in the same complex (Fig. 5C). Most importantly, the endogenous STK38 was found to form a complex with DIAPH3 in the immunoprecipitation assay (Fig. 5D).
3.6. DIAPH3 rescued the inhibitory effects of STK38 To examine the functional link between DIAPH3 and STK38, we simultaneously over-expressed Flag-DIAPH3 and HA-STK38 in SPC-A1 cells (Fig. 7A). Overexpression of DIAPH3 effectively abolished the inhibitory effects of STK38 on the anchorage-independent growth of SPC-A-1 cells (Fig. 7B). Consistently, Overexpression of DIAPH3 effectively rescued the MEK kinase protein level (Fig. 7C). To understand the molecular mechanism, the interaction between STK38 and MEK kinase 6
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Fig. 5. DIAPH3 interacted with STK38. (A) Silver staining and mass spectrum were performed to screen the binding protein of DIAPH3. 293 T cells were transfected with Flag-DIAPH3 plasmids. 48 h later, the immunoprecipitation was performed using the anti-Flag antibody. The immunoprecipitates were separated with 10% SDSPAGE, and the differential band at about 60 KD (arrow) was cut for mass spectrum analysis. (B) GST pull-down assay was performed to examine the interaction between DIAPH3 and the fusion protein GST-STK38 in SPC-A-1 cells. (C) Immunoprecipitation assay was performed to examine the interaction between DIAPH3 and STK38. Flag-DIAPH3 and HA-STK38 plasmids were transfected into SPC-A-1 cells. 48 h later, the immunoprecipitation was performed using the anti-HA antibody. (D) Immunoprecipitation assay was performed to examine the interaction between endogenous DIAPH3 and STK38 in SPC-A-1 cells.
4. Discussion
was evaluated. Interestingly, DIAPH3 disrupted the interaction between STK38 and MEK kinase (Fig. 7D). Moreover, the rescue effects of DIAPH3 on the inhibitory roles of STK38 could be attenuated by the ERK inhibitor, LY3214996 (Fig. 7B), suggesting that DIAPH3 rescued the inhibitory effects of STK38 and activated ERK signaling.
Aberrant activation of MEK-ERK signaling is very common in the lung cancer [20]. However, how the MEK-ERK signaling is regulated does not fully understood. In this study, we have found that DIAPH3 upregulated the MEKK protein level by inhibiting the interaction between
Fig. 6. STK38 inhibited the growth of LUAD cells. (A) Mining the GEPIA database revealed that no difference of STK38 expression between the LUAD tissues and adjacent normal tissues. (B) Mining the GEPIA database revealed that no correlation between STK38 expression and the survival of patients. (C) STK38 hindered the growth of SPC-A-1 in the MTT assay. (D) STK38 hindered the anchorage-independent growth of A549 and SPC-A-1 cells in the soft agar assay. (E) STK38 knockdown up-regulated the MEKK protein level. **, P < 0.01. 7
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Fig. 7. DIAPH3 attenuated the interaction between STK38 and MEKK. (A) Simultaneously expression of Flag-DIAPH3 and HA-STK38 in SPC-A-1 cells was examined using western blot. (B) The rescue effects of DIAPH3 on the anchorage-independent growth of SPC-A-1 cells were examined using soft agar. The ERK inhibitor, LY3214996, was used to examine the roles of ERK signaling. (C) The rescue effects of DIAPH3 on the expression of MEKK were examined using western blot. (D) DIAPH3 disrupted the interaction between MEKK and STK38. **, P < 0.01.
the up-regulation of DIAPH3, the interaction between the STK38-MEKK was attenuated, thus the MEK/ERK signaling was activated. In summary, we have demonstrated the oncogenic roles of DIAPH3 in lung cancer by interacting with STK38 in this study, and suggested that DIAPH3 might be a therapeutic target.
STK38 and MEK, thus promoted the tumorigenicity of lung cancer cells. These results clearly demonstrated the oncogenic roles of DIAPH3 in the lung cancer. Several observations were provocative for a few reasons. First, the up-regulation of DIAPH3 was only observed in the lung adenocarcinoma. One of the good explanations for this might be that DIAPH3 expression was negatively regulated by P53. This hypothesis was based on two aspects: 1. The Ad-Cre; P53f/f; KrasG12D lung adenocarcinoma mice showed higher DIAPH3 protein levels; 2. The loss-of-function mutation of P53 is very common in lung adenocarcinoma than the lung squamous cell carcinoma. Based on these observations, DIAPH3 might be a bio-marker for lung adenocarcinoma. Further, DIAPH3 promoted the anchorage-independent growth of lung cancer cells. It has been reported that DIAPH3 regulated the dynamics of cytoskeleton and cell motility. Although we did not examine the functions of DIAPH3 in the migration and metastasis of lung cancer cells, it could not rule out the possibility that DIAPH3 regulated the migration and metastasis of lung cancer cells. One of the most interesting findings of this study was the attenuated interaction between STK38 and MEKK by DIAPH3. It has been known that STK38 interacted with MEKK and inhibited the activation of MEK/ ERK signaling [7]. In the normal cells, the interaction between STK38DIAPH3 and STK38-MEKK reached the balance. In cancer cells, due to
Ethics approval and consent to participate All experimental protocols were approved by Shanghai Jiaotong University institutional committee. Informed consent was obtained from all subjects. The study was reviewed and approved by the China national institutional animal care and use committee.
Consent for publication All authors agreed on the manuscript.
Declaration of competing interest The authors declare that they have no competing interests. There is no conflict of interest. 8
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Appendix A. Supplementary data [11]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yexcr.2019.111662.
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References [13]
[1] M. Cully, Cancer: closing the door on KRAS-mutant lung cancer, Nature reviews, Drug Discov. 15 (2016) 747. [2] R. Hansen, U. Peters, A. Babbar, Y. Chen, J. Feng, M.R. Janes, L.S. Li, P. Ren, Y. Liu, P.P. Zarrinkar, The reactivity-driven biochemical mechanism of covalent KRAS (G12C) inhibitors, Nat. Struct. Mol. Biol. 25 (2018) 454–462. [3] M.P. Patricelli, M.R. Janes, L.S. Li, R. Hansen, U. Peters, L.V. Kessler, Y. Chen, J.M. Kucharski, J. Feng, T. Ely, J.H. Chen, S.J. Firdaus, A. Babbar, P. Ren, Y. Liu, Selective inhibition of oncogenic KRAS output with small molecules targeting the inactive state, Cancer Discov. 6 (2016) 316–329. [4] K. Nawaz, R.M. Webster, The non-small-cell lung cancer drug market, Nature reviews, Drug Discov. 15 (2016) 229–230. [5] C. Joffre, P. Codogno, M. Fanto, A. Hergovich, J. Camonis, STK38 at the crossroad between autophagy and apoptosis, Autophagy 12 (2016) 594–595. [6] D.D. Shi, H. Shi, D. Lu, R. Li, Y. Zhang, J. Zhang, NDR1/STK38 potentiates NFkappaB activation by its kinase activity, Cell Biochem. Funct. 30 (2012) 664–670. [7] N. Xiao, H. Li, W. Yu, C. Gu, H. Fang, Y. Peng, H. Mao, Y. Fang, W. Ni, M. Yao, SUMO-specific protease 2 (SENP2) suppresses keratinocyte migration by targeting NDR1 for de-SUMOylation, FASEB J. : Off. Publ. Fed. Am. Soc. Exp. Biol. 33 (2019) 163–174. [8] C.D. Ji, Y.X. Wang, D.F. Xiang, Q. Liu, Z.H. Zhou, F. Qian, L. Yang, Y. Ren, W. Cui, S.L. Xu, X.L. Zhao, X. Zhang, Y. Wang, P. Zhang, J.M. Wang, Y.H. Cui, X.W. Bian, Kir2.1 interaction with Stk38 promotes invasion and metastasis of human gastric cancer by enhancing MEKK2-MEK1/2-ERK1/2 signaling, Cancer Res. 78 (2018) 3041–3053. [9] M. Katoh, M. Katoh, Identification and characterization of human DIAPH3 gene in silico, Int. J. Mol. Med. 13 (2004) 473–478. [10] F. Calvo, N. Ege, A. Grande-Garcia, S. Hooper, R.P. Jenkins, S.I. Chaudhry, K. Harrington, P. Williamson, E. Moeendarbary, G. Charras, E. Sahai, Mechanotransduction and YAP-dependent matrix remodelling is required for the
[14]
[15]
[16]
[17]
[18]
[19]
[20]
9
generation and maintenance of cancer-associated fibroblasts, Nat. Cell Biol. 15 (2013) 637–646. L. Dong, Z. Li, L. Xue, G. Li, C. Zhang, Z. Cai, H. Li, R. Guo, DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling, Mol. Cell. Biochem. 438 (2018) 183–190. M.H. Hager, S. Morley, D.R. Bielenberg, S. Gao, M. Morello, I.N. Holcomb, W. Liu, G. Mouneimne, F. Demichelis, J. Kim, K.R. Solomon, R.M. Adam, W.B. Isaacs, H.N. Higgs, R.L. Vessella, D. Di Vizio, M.R. Freeman, DIAPH3 governs the cellular transition to the amoeboid tumour phenotype, EMBO Mol. Med. 4 (2012) 743–760. S.H. Bae, J.I. Baek, J.D. Lee, M.H. Song, T.J. Kwon, S.K. Oh, J.Y. Jeong, J.Y. Choi, K.Y. Lee, U.K. Kim, Genetic analysis of auditory neuropathy spectrum disorder in the Korean population, Gene 522 (2013) 65–69. G.M. Carvalho, P.Z. Ramos, A.M. Castilho, A.C. Guimaraes, E.L. Sartorato, Molecular study of patients with auditory neuropathy, Mol. Med. Rep. 14 (2016) 481–490. J.A. Vorstman, E. van Daalen, G.R. Jalali, E.R. Schmidt, R.J. Pasterkamp, M. de Jonge, E.A. Hennekam, E. Janson, W.G. Staal, B. van der Zwaag, J.P. Burbach, R.S. Kahn, B.S. Emanuel, H. van Engeland, R.A. Ophoff, A double hit implicates DIAPH3 as an autism risk gene, Mol. Psychiatry 16 (2011) 442–451. R. Lang-Roth, E. Fischer-Krall, C. Kornblum, G. Nurnberg, D. Meschede, I. Goebel, P. Nurnberg, D. Beutner, C. Kubisch, M. Walger, A.E. Volk, AUNA2: a novel type of non-syndromic slowly progressive auditory synaptopathy/auditory neuropathy with autosomal-dominant inheritance, Audiol. Neuro. Otol. 22 (2017) 30–40. M. Reis-Sobreiro, J.F. Chen, T. Novitskaya, S. You, S. Morley, K. Steadman, N.K. Gill, A. Eskaros, M. Rotinen, C.Y. Chu, L.W.K. Chung, H. Tanaka, W. Yang, B.S. Knudsen, H.R. Tseng, A.C. Rowat, E.M. Posadas, A. Zijlstra, D. Di Vizio, M.R. Freeman, Emerin deregulation links nuclear shape instability to metastatic potential, Cancer Res. 78 (2018) 6086–6097. J. Stastna, X. Pan, H. Wang, A. Kollmannsperger, S. Kutscheidt, V. Lohmann, R. Grosse, O.T. Fackler, Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation, Cell Res. 22 (2012) 728–745. J. Jiang, Diaphanous-related formin-3 overexpression inhibits the migration and invasion of triple-negative breast cancer by inhibiting RhoA-GTP expression, Biomed. pharmacother. 94 (2017) 439–445. C. Ferte, F. Andre, J.C. Soria, Molecular circuits of solid tumors: prognostic and predictive tools for bedside use, Nature reviews, Clin. Oncol. 7 (2010) 367–380.