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Amphiregulin (AR) is derived from a membrane-anchored form (proAR) by ectodomain shedding, and is a ligand that activates epidermal growth factor receptor (EGFR). We have recently shown that proAR translocates from the plasma membrane to the nucleus after truncation of 11 amino acids at the C-terminus, which is independent of the conventional EGFR signaling pathway. Although proAR immunoreactivity has reportedly been detected in the nucleus of cancer cells, its biological meaning has never been investigated. This study was performed to investigate the roles of proAR nuclear translocation in human gastric cancer. We constructed proAR truncated 11 amino acids at the C-terminus (proARΔC11) that spontaneously translocates to the nucleus, and established proARΔC11-expression regulatable gastric cancer cells (MKN45, MKN28) using the tet-off system. Using these cells, we found that proAR nuclear translocation significantly induced chemoresistance in vitro and in vivo. Analyzing the relationship between immunoreactive localization of proAR and the clinical outcome for 46 advanced gastric cancer cases treated with chemotherapy, median survival time was 311 days in 16 patients with AR-positive staining in the nucleus and 387 days in 30 patients with AR-negative staining (P < 0.05). The present study demonstrates that proAR nuclear translocation increases resistance to anti-cancer drugs, which might be associated with poor prognosis in human gastric cancer. (Cancer Sci 2012; 103: 708–715)
All of the epidermal growth factor (EGF) receptor (EGFR) ligands are cleaved from membrane-anchored precursors (pro-forms) at the plasma membrane in a process termed “ectodomain shedding.” The ligand binding to EGFR induces activation of intracellular signaling cascades implicated in the regulation of a wide variety of cellular processes, including growth, differentiation, apoptosis, adhesion and migration. Deregulated expression and activation of EGFR is now widely accepted to play a role in cancer progression. As a result, EGFR has become a therapeutic target and many EGFR inhibitors are in clinical use.
Epidermal growth factor receptor is highly expressed in 33–74% of gastric cancers, and elevated levels have been reported as an independent indicator of poor prognosis.[3-5] Furthermore, increased expression of EGFR ligands, such as transforming growth factor (TGF)-α, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR), is associated with a poor clinical prognosis in many cancers, including gastric cancer.[6, 7] In addition, the EGFR and EGFR ligand gene families have been associated with growth regulation and gastric wall invasion in vitro.
Despite aberrant enhancement of both EGFR and EGFR ligand expression in gastric cancers, EGFR-targeted therapeutics does not clinically show sufficient benefit against gastric cancer. These results suggest that other signal pathways might be present in the EGFR–ligand signal cascade besides the conventional signal that activates EGFR by binding with ligands. We have previously demonstrated a new function of proHB-EGF that is not associated with the conventional EGFR signaling pathway. In this mechanism, the carboxyl-terminal fragment (HB-EGF-CTF) generated by ectodomain shedding of proHB-EGF translocates from the plasma membrane into the inner nuclear membrane and regulates transcriptional repressors.[9-11] Moreover, we have reported that HB-EGF-CTF might offer a new molecular target for treating gastric cancer. However, mechanisms besides the EGFR-binding cascade are little understood in terms of other EGFR ligands.
Amphiregulin is also a member of the EGF family, and its increased expression has been reported in many epithelial cancers, including gastric cancer.[13-18] AR is considered to be a new serological marker for breast cancer and colorectal cancer.[19-21] In non-small cell lung cancer, AR is associated with poor prognosis. Therefore, AR has been recognized as one of the most important factors in the EGF family for cancer therapy. Many studies have detected AR immunoreactivity in human gastric cancer tissues, in the cytoplasm and nucleus of tumor cells.[14, 23] However, the biological function of AR in the cell nucleus has long been unclear. We recently reported that a significant amount of the precursor of AR (proAR) translocates from the plasma membrane to the nuclear membrane and that truncation of the proAR C-terminal tail is required for the nuclear translocation. ProAR that translocates to the nucleus suppresses global transcription (Fig. 1A). However, the mechanisms through which proAR nuclear translocation affects human cancer remain unclear. We presumed that proAR nuclear translocation, along with proHB-EGF, might have important effects on progression and chemotherapy for gastric cancer. To investigate the specific role of proAR localized to the nucleus, we require a good system to induce spontaneous translocation of proAR to the nucleus, because we have to exclude side effects of shedding stimulants that has multiple effects on cells. Therefore, we constructed a mutant of proAR, proARΔC11, which lacks C-terminal 11 amino acids and spontaneously translocates to the nucleus without the production of released AR, and established human gastric cancer cells with inducible proARΔC11 expression, using a tetracycline (tet)-regulating system, the tet-off system.
Figure 1. (A) Amphiregulin (AR) is expressed on the plasma membrane as proAR, consisting of a predicted 252 amino acids. ProAR primarily localizes at the plasma membrane. Stimulation induces partial proAR-ectodomain shedding, resulting in production of soluble AR and the carboxyl-terminal fragment of proAR (AR-CTF). In addition, stimulation causes endocytosis of the residual proAR. During translocation to the nucleus, proAR is truncated to the 11 amino acids at the C-terminus (proARΔC11), and proARΔC11 is targeted to the endoplasmic reticulum (ER). ER-localized proARΔC11 subsequently diffuses or is actively transported to the nucleus. (B) Schematic presentation of V5-tag inserted proAR and its deletion mutants. V5-tagged proAR protein was termed wt-proAR, and V5-tagged proARΔC11 protein was proARΔC11. MKN45 lines transfected with the tet-off system for consistent and conditional induction of wt-proAR and proARΔC11 were named “MKN45/wt-proAR” and “MKN45/proARΔC11,” respectively. (C) Western blot analysis of proAR and AR-CTF expression in MKN45, MKN45/wt-proAR and MKN45/proARΔC11. Each lane contained 100 μg of protein. The concentration of 12-0-tertadecanoylphorbor-13-acetate (TPA) was 200 nmol/L to induce ectodomain shedding. We used anti-V5 antibody (V5 Ab) to recognize the cytoplasmic region of wt-proAR and proARΔC11, and anti-AR antibody (AR-N Ab) to recognize the proAR ectodomain. Loading control comprised β-actin. (D) Immunoprecipitation and western blot analysis of MKN45/wt-proAR and MKN45/proARΔC11. The concentration of TPA was 200 nmol/L. Cell lysates were immunoprecipitated with anti-lamin A/C antibody (Lamin A/C Ab), and precipitated proteins were analyzed by western blotting using AR-N Ab and Lamin A/C Ab. The cell lysates were analyzed using Lamin A/C Ab. (E) Intracellular localization of proAR after TPA-inducible processing in MKN45/wt-proAR and MKN45/proARΔC11 under immunofluorescence microscopy. The concentration of TPA was 200 nmol/L. Nucleus stained blue with DAPI, AR stained green with AR-N Ab and AR-CTF stained green with V5 Ab.
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We investigate the biological behavior of proARΔC11 nuclear translocation using this system and present the novel finding that proAR nuclear translocation increases chemoresistance and represents a new target for gastric cancer therapy.
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Epidermal growth factor receptor and EGFR ligands have been well studied as molecular targets for cancer therapy.[28-30] AR has been characterized as a growth factor involved in cell proliferation and differentiation, AR is recognized as an important factor affecting cancer therapy.[14-22] We recently reported that a significant amount of proAR is internalized without ectodomain shedding in response to shedding stimuli and translocates to the nucleus. This signal transduction induces heterochromatin formation and suppression of global transcription. However, the relationship between proAR nuclear localization and cancer has not yet been reported. Therefore, the present work focuses on proAR nuclear translocation as a new molecular target for gastric cancer. In completing this study, we successfully established gastric cancer cell lines with tet-regulated inducible proAR nuclear translocation, MKN45/proARΔC11. We were thus able to selectively analyze the basic effects of proAR nuclear translocation, both in vitro and in vivo.
Our study revealed that spontaneous nuclear translocation of proARΔC11 significantly slowed proliferation and induced chemoresistance to three anti-cancer drugs, CDDP, PTX and 5-FU, in vitro and in vivo. Overexpression of wt-proAR enhanced cell growth, which is explained by the production of released AR and EGFR activation because constitutive shedding of proAR in MKN45/wt-proAR was observed (Fig. 1C). However, chemoresistance of MKN45/wt-proAR to anti-cancer drugs did not show significant correlation, which might be explained by the different extent of nuclear translocation of proAR.
Furthermore, we immunohistochemically investigated AR expression on specimens of human gastric cancers. Our data indicate that the AR (+) group experienced significantly poorer prognosis. Although significant differences were not noted, probably due to the small sample size, response rates tended to be lower in the AR (+) group than in the AR (−) group. These results supported the present data shown in vitro and in vivo. Although many previous reports have shown AR immunoreactivity in cancers, including gastric cancer, to be detected in both the cytoplasm and the nuclei of tumor cells, the significance of AR localization in the nucleus has not been clarified.[1, 31-33] In the present study, AR was expressed in both the nucleus and the cytoplasm in all AR (+) cases. AR immunoreactivity in the nucleus must show translocated proAR. These results suggest that proAR nuclear localization might play a crucial role in the determination of some tumor properties.
We have previously reported that HB-EGF-CTF translocates from the plasma membrane to the nucleus and regulates the cell cycle by abrogation of transcriptional repressors.[9, 10] Moreover, we have shown that it is important for gastric cancer therapy to inhibit not only the conventional EGFR signal pathway, but also HB-EGF-CTF nuclear translocation. The present study shows that proAR nuclear translocation might offer a new molecular target for gastric cancer progression, along with HB-EGF. The present study also indicates that nuclear translocation as well as overexpression of proAR could be critical for molecular diagnosis of gastric cancer. We found some squamous cell carcinoma cell lines presenting spontaneous nuclear translocation of endogenous proAR (Shigeki Higashiyama, unpublished observation). Although we have not yet elucidated the existence of mutant gene expressing proARΔC11 in living cells, C-terminal modification of proAR, such as proteolytic cleavage, and its mediating factors might be good molecular targets for diagnosis and therapy.
Cancer cells can acquire resistance to chemotherapy by a range of mechanisms. In general, chemoresistance is attributed to mutation or overexpression of the drug target, inactivation of the drug, or elimination of the drug from the cell. However, these considerations cannot confer full resistance to multiple anti-cancer drugs with different mechanisms of action. We have not yet elucidated how proAR nuclear translocation induces chemoresistance. Further investigation remains necessary, as the detailed mechanisms are not yet fully understood, and clarifying these mechanisms would certainly result in the development of new cancer therapies.
In conclusion, this study is the first report to show basic and clinical behavior of proAR nuclear translocation. Our results indicate that proAR nuclear translocation induces chemoresistance to anti-cancer drugs and might be associated with poor prognosis in human gastric cancer. ProAR nuclear translocation might offer a prognostic marker and a new molecular target for gastric cancer therapy.