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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Genetic crossing experiments were performed between tuberous sclerosis-2 (Tsc2) KO and expressed in renal carcinoma (Erc) KO mice to analyze the function of the Erc/mesothelin gene in renal carcinogenesis. We found the number and size of renal tumors were significantly less in Tsc2+/−;Erc−/− mice than in Tsc2+/−;Erc+/+ and Tsc2+/−;Erc+/− mice. Tumors from Tsc2+/−;Erc−/− mice exhibited reduced cell proliferation and increased apoptosis, as determined by proliferating cell nuclear antigen (Ki67) and TUNEL analysis, respectively. Adhesion to collagen-coated plates in vitro was enhanced in Erc-restored cells and decreased in Erc-suppressed cells with siRNA. Tumor formation by Tsc2-deficient cells in nude mice was remarkably suppressed by stable knockdown of Erc with shRNA. Western blot analysis showed that the phosphorylation of focal adhesion kinase, Akt and signal transducer and activator of transcription protein 3 were weaker in Erc-deficient/suppressed cells compared with Erc-expressed cells. These results indicate that deficiency of the Erc/mesothelin gene ameliorates renal carcinogenesis in Tsc2 KO mice and inhibits the phosphorylation of several kinases of cell adhesion mechanism. This suggests that Erc/mesothelin may have an important role in the promotion and/or maintenance of carcinogenesis by influencing cell-substrate adhesion via the integrin-related signal pathway. (Cancer Sci 2011; 102: 720–727)

Expressed in renal carcinoma (Erc) was identified as an inducible gene during renal carcinogenesis in the Eker (tuberous sclerosis-2 [Tsc2] mutant) rat. The background of this research originates from our studies of the mechanism of multi-step carcinogenesis in an animal model involving the Tsc2 mutant gene using the Eker rat.(1–4) Development of hereditary renal carcinomas in the Eker rat is initiated by a somatic second hit(5) of the Tsc2 gene. To elucidate the “steps” involved in Tsc2-deficiency, genes induced during renal carcinogenesis were cloned in the Eker rat and Erc was identified as a novel gene. Subsequently, it was revealed that Erc is a homologue of the human mesothelin gene.(6) Also, Erc protein is a homologue of a 31-kDa megakaryocyte potentiating factor (MPF), which can stimulate the megakaryocyte colony-forming activity of murine interleukin-3 in mouse bone marrow cell culture. Moreover, Erc protein was also cloned as an antigenic mesothelin for the monoclonal antibody K1 raised against ovarian cancer.(7–9)

Erc/mesothelin protein is a glycosyl phosphatidylinositol (GPI)-anchored membrane glycoprotein that is expressed in normal mesothelial cells. It is also highly expressed in several species of malignant tumors, such as mesothelioma as well as ovarian and pancreatic cancers.(10–13) Its primary product, a 71-kDa precursor of protein, can be physiologically cleaved by a furin-like protease into two fragments. A 31-kDa amino-terminal fragment (MPF, described hereafter as N-Erc/mesothelin) is released into the extra-cellular fluids, while a 40-kDa carboxy-terminal fragment (C-Erc/mesothelin) remains in the cell membranes.(10–16) Soluble N-Erc/mesothelin in serum is already being utilized as a diagnostic tumor marker(14–16) and anti-C-Erc/mesothelin immunotoxin therapy has been reported to be effective for mesothelioma and some C-Erc/mesothelin-expressing cancers.(10) Several in vitro studies have suggested that activation of cancer-associated signaling pathways increases Erc/mesothelin expression, and Erc/mesothelin may play a role in tumor adhesion, dissemination, metastasis and resistance against cell death.(9,10,17–22) However, mutant mice in which both copies of the mesothelin gene were inactivated showed no detectable abnormalities when compared with wild-type littermates.(23) Thus, it is conceivable that Erc/mesothelin may have a specific role in carcinogenesis as well as pathogenesis.

The Erc/mesothelin gene, described hereafter as Erc, is also highly expressed in renal tumor cells from Tsc2 KO heterozygous mutant (Tsc2+/−) mice, that develop hereditary renal tumors presenting as cysts, cyst-adenomas, and solid adenomas that histologically resemble those in the Eker rat.(24)

In this study, Tsc2+/−;Erc−/− double mutant mice were generated through meiotic recombination and several renal tumor cell lines were established. The phenotypes of the Tsc2 KO mice with or without Erc expression were compared and functions of the Erc gene in carcinogenesis were analyzed in vivo and in vitro. We report here that the development of renal tumors was significantly reduced in Tsc2+/−;Erc−/− mice, as compared to Tsc2+/− (Erc WT) or Tsc2+/−;Erc+/− mice and the several phosphorylation events mediated by integrin and the mammalian target of rapamycin (mTOR) were disrupted in Tsc2;Erc double deficient renal tumor cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Gene targeting and generation of Erc knockout mouse and crossing with Tsc2 knockout mouse.  Genomic DNA clones covering Erc were prepared from a mouse genomic DNA library and used for construction (see Data S1 and Fig. S1 for details).

Tumor measurement and tissue preparation.  Mice were sacrificed at 18 months of age. The visible tumors on the renal surface were counted and measured with a caliper for length and width. The size of a tumor was defined to be the tumor’s mean diameter: (length [mm] + width [mm])/2. Mice were divided into three groups according to the size of their largest tumor: small (<3 mm), large (3–10 mm) and extra-large (>10 mm). Tissues were fixed in 10% neutral formalin and paraffin sections (3 μm each) were prepared for examination.

Cell adhesion assay.  The assay was performed according to a method described previously(25) with minor modifications and with type I collagen-coated 24-well plates (Iwaki, Tokyo, Japan). Briefly, after blocking nonspecific adhesion with 1% bovine serum albumin in PBS, 1 × 105 cells suspended in 1.0 mL of 10% FCS/RPMI-1640 were added to each well and the cells were allowed to adhere for 1 h at 37°C in a 5% CO2 incubator. After washing with PBS, the remaining cells were stained with 0.5% crystal violet in 20% methanol for 30 min and then washed away with water. The stained cells were solubilized in 20% acetic acid and the absorbance of the solution was read in a microplate spectrophotometer (Benchmark Plus; Bio-Rad, CA, USA) at 595 nm. Three independent experiments were performed in quadruplicate.

Transplantation assay.  BALB/c nude mice were injected subcutaneously with 5 × 106 tumor cells in 100 μL of serum-free medium. After tumors appeared, the tumors were measured weekly with a caliper for length, width and height and the volume was calculated using the following formula: tumor volume (mm3) = length (mm) × width (mm) × height (mm)/2.

Statistical analysis.  All discrete values, expressed as mean ± SEM, were analyzed using Student’s t-test. P-values of <0.05 were considered statistically significant.

See Supporting Information (Data S1) for additional methods.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Establishment of Tsc2;Erc double knockout mice. Trans-compound double heterozygous mutant (Tsc2+/− and Erc+/−) male mice were mated with C57BL/6J (WT) females (Fig. 1a). Of the 109 offspring, there was a single mouse carrying both Tsc2 and Erc mutations that is a cis-compound double heterozygous mutant (Fig. 1b, white star), provisionally designated Erc109. Tsc2+/−;Erc−/− mice (Fig. 1b, black stars) were established by mating Erc109 (Tsc2+/−;Erc+/−) with Erc KO mice (Fig. 1a). Absence of Erc protein in Tsc2+/−;Erc−/− mice was confirmed immunohistochemical staining of the lung mesothelium (Fig. 2a) using anti-mouse C-Erc/mesothelin rabbit polyclonal antibody (Fig. S2).

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Figure 1.  Generation of tuberous sclerosis-2 (Tsc2); expressed in renal carcinoma (Erc) double KO mouse. (a) Outline of intercrosses. Only Erc-deficient mouse is denoted after second mating. (b) Genotyping of genomic DNA by PCR. Representative results of offspring from first cross (left panels) and second cross (right panels) are shown. Upper and lower panels show genotypes of Tsc2 and Erc, white and black stars indicate the Tsc2+/−;Erc+/− (cis-compound heterozygous) mutant and the Tsc2+/−;Erc−/− mutant, respectively.

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Figure 2.  Reduction of the number and size of renal tumors in expressed in renal carcinoma (Erc)-deficient mice. (a) Immunohistochemical staining of anti C-Erc/mesothelin show the positive reactions (arrows) in lung mesothelium of tuberous sclerosis-2 (Tsc2)+/− (Erc WT) and Tsc2+/−;Erc+/− mice, but not in Tsc2+/−;Erc−/− mice (arrowheads). Scale bars = 20 μm. (b) Representative macroscopic findings of the renal tumors (arrows) in 18-month-old mice. Scale bars = 3 mm. (c) H&E staining of sections of renal tumors of above mice. Arrows and arrowheads indicate tumors and normal tissues, respectively. Scale bars = 40 μm. (d) The number of renal tumors was evaluated and expressed as average number per mouse. Values are means ± SEM; **< 0.01. Number of mice measured in each group is shown in columns. (e) The number of large-size (≥3 mm) tumors was selected from (d) and expressed as average number per mouse. Values are means ± SEM; **< 0.01. The number of mice measured in each group is the same as (d). (f) The mice were categorized into three groups according to the size (mean diameter) of the largest tumor that the mouse harbored. Points = the largest tumor of each mouse; ***< 0.001. The number of mice measured in each group is the same as (d). The large-size and extra-large-size tumor numbers in the Erc−/− mice are significantly less when compared with the other mouse strains.

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Reduced renal tumor development in Tsc2+/−;Erc−/− mice.  Renal tumor development was examined in Tsc2+/− (Erc WT), Tsc2+/−;Erc+/− and Tsc2+/−;Erc−/− mice (Fig. 2b–f). Renal tumors were developed in all of the above mice at 18 months of age (Fig. 2b,c,f). However the number (Fig. 2d) and size (mean diameter; Fig. 2b,e,f) of visible tumors on the renal surface were significantly reduced in the Tsc2+/−;Erc−/− mice. In Tsc2+/− as well as Tsc2+/−;Erc+/− mice, frequent development of tumors of large-size (≥3 mm) were observed in both females and males. There were 59.3% (16 of 27) female and 58.3% (14 of 24) male Tsc2+/− mice and 58.6% (17 of 29) female and 57.1% (16 of 28) male Tsc2+/−;Erc+/− mice that developed large-size tumors. In contrast, only two of 20 females (10.0%) and one of 15 males (6.7%) of Tsc2+/−;Erc−/− mice showed such large-size tumors (Fig. 2f), although they commonly exhibited carcinogenesis. Moreover, extra-large-size (>10 mm) tumors were seen in Tsc2+/− and Tsc2+/−;Erc+/− mice (11 and 13 cases, respectively) but were not found in Tsc2+/−;Erc−/− mice (Fig. 2b,f). These observations suggest that the progression of renal tumors in Tsc2 mutant mice was suppressed by Erc-deficiency.

Decreased proliferation in renal tumors from Tsc2+/−;Erc−/− mice.  To elucidate the cellular basis for the effects of Erc-deficient on proliferation and apoptosis of renal tumors, the paraffin-embedded renal tumor sections were stained with anti-mouse Ki67 (a proliferating cell nuclear antigen) by immunohistochemistry and TUNEL analysis, respectively (Fig. 3). Tumors derived from Tsc2+/−;Erc−/− mice not only were significantly reduced tumor cell proliferation (Fig. 3a) but also showed increased apoptosis (Fig. 3b) compared with tumors from Tsc2+/−; Erc−/− mice although Erc-deficient tumors exhibited a mild increase in apoptosis status (Fig. 3c).

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Figure 3.  Expressed in renal carcinoma (Erc)-deficient decreases tumor growth by impairing cell proliferation. (a) Proliferation was assessed by immunohistochemistry staining on sections of paraffin-embedded renal tumors of the indicated genotype with Ki67 antibody. Positive cells appear brown (arrows). Scale bars = 40 μm. (b) TUNEL-stained sections of paraffin-embedded renal tumors of the indicated genotype. Apoptotic cells appear brown (arrows). Scale bars = 40 μm. (c) The number of Ki67-positive cells (left panel) or TUNEL-positive cells (right panel) per microscopic field was evaluated as described in the Data S1. Values are means (= 30 images per genotype) ± SEM; **< 0.01; *< 0.05.

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Positive effects of Erc on collagen-mediated cell-substrate adhesion in renal tumor cell lines.  To conduct an in vitro functional analysis of Erc, renal tumor cell lines were established from Tsc2+/−;Erc−/− mice. Then, Erc expression was restored in one of the established cell lines (DE42L-T1-9) by stable transduction of an Erc expression vector and the expression of Erc protein was shown by Western blot (Fig. 4a). Erc-restored cells (T1-9Ep10 and T1-9Ep13) were found to be more competent to adhere on the collagen-coated plates than Erc-deficient (parental and empty-vector) cells (Fig. 4b). To ascertain that the increased adhesion was due to the function of Erc, the expression of Erc in Erc-restored cells was re-suppressed by RNAi and the effect of Erc-suppression was verified by RT-PCR and Western blot (Fig. 4c). The adhesion of these cells on collagen-coated plates was significantly reduced by the suppression of Erc (Fig. 4d), confirming that Erc positively regulates cell-substrate adhesion.

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Figure 4.  Expressed in renal carcinoma (Erc)-expression enhanced cell adhesion to collagen-coated plates. (a) Western blot analysis showed the level of N-Erc expression in the cell lines used for cell adhesion assay. Name of each cell used for further analysis is shown. (b) The adhesion was enhanced in stable Erc-restored cells (T1-9Ep10 and T1-9EP13) compared with Erc-deficient parental cell line (DE42L-T1-9) and empty-vector cells (T1-9puro1 and T1-9puro4). Values are means ± SEM; ***< 0.001. Three independent experiments were performed in quadruplicate. (c) RT-PCR (Erc and β-Actin) and Western blot analysis (N-Erc) showed the effects of Erc-suppression when treated with Erc siRNA in Erc-restored cells. (d) In contrast to results in (b), the adhesion was decreased in Erc-restored cells treated with siRNA. Values are means ± SEM; ***< 0.001; **< 0.01. Three independent experiments were performed in quadruplicate.

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Suppression of tumorigenesis with Erc-suppression in Tsc2-deficient renal tumor cells. Erc-suppressed cells were established by stable expression of shRNA from the MKOC1-277 cell line that is a Tsc2-deficient mouse renal tumor cell line with highly expressed Erc (Fig. 5a). When in vivo tumorigenicity was examined by subcutaneous injections of cells into nude mice, tumors generated from the Erc-suppressed cells were smaller and paler than those from the control shRNA cells that showed robust tumorigenesis (Fig. 5b,c). Conversely, when Erc-restored cells were assayed, they exhibited more vigorous growth compared with Erc-deficient (empty-vector) cells (Fig. S3). These data suggest that Erc exerts a positive effect on tumorigenicity.

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Figure 5.  Expressed in renal carcinoma (Erc)-deficiency suppressed tumorigenicity of renal tumor cells transplanted in nude mice. (a) Western blot analysis showed the level of N-Erc expression in the stable Erc-suppressed cell lines. Black and white stars indicate the cells used for tumorigenicity assay shown below. (b) The tumor volumes were suppressed in stable Erc-suppressed (MKOCshE15, black circles) cells compared with control-shRNA (MKOCshC8, white circles) cells after implantation into nude mice. Values are means ± SEM (= 6); **< 0.01; *< 0.05. (c) Macroscopic appearance of tumors was shown at 8 weeks after implantation. Tumor areas are marked with hexagons. Scale bars = 10 mm.

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Modulation of integrin-related signaling by Erc expression.  Integrin β1 is a major subunit of collagen receptors(26) and is required for collagen-mediated proliferation of cancer cells.(27–29) Signals from the integrin complex are transmitted through the phosphorylation of focal adhesion kinase (FAK).(30–32) To determine if Erc expression affects cell adhesion through integrin-related signaling, the expression of integrin β1 and the phosphorylated status of downstream molecules were compared among the indicated cell lines (Fig. 6). As previously reported, two major bands were observed in Western blots of integrin β1,(33–37) namely a partially glycosylated 115 kDa precursor and a fully glycosylated 135 kDa mature form. These bands were disappeared or abolished and a band of core peptide (86 kDa) was appeared upon mild (2 μg/mL) tunicamycin (a N-glycosylation inhibitor) treatment, confirming the characteristics of integrin β1 (Fig. S4). Mature integrin β1 was dominant in Erc-deficient (parental and empty-vector) cells, while the expression of integrin β1 shifted to the precursor in Erc-restored cells (Fig. 6a). Reciprocally, levels of the precursor integrin β1 were decreased in Erc-suppressed cells compared with Erc-expressed (parental and control shRNA) cells (Fig. 6b). Although direct evidence has not yet been obtained, it is plausible that the expression of mature integrin β1 may be regulated by feedback from cell adhesion machinery regulated by Erc.

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Figure 6.  Expressed in renal carcinoma (Erc) expression affects integrin-related signal. Western blot analysis was performed with indicated antibodies; the concentrated culture supernatant lysates were used for N-Erc and whole-cell lysates were used for other antibodies. (a) The stable Erc-restored cells (central two lanes) increased the precursor integrin β1 (lower-band) and the level of phosphorylation of focal adhesion kinase (FAK), Akt, S6K, rpS6 and Stat3 compared with Erc-deficient parental cell line (left lane) and the empty-vector cell (right lane). (b) The stable Erc-suppressed cells (right two lanes) decreased the precursor integrin β1 (lower-band) and the level of phosphorylation of FAK, Akt, S6K, rpS6 and Stat3 compared with Erc-express (WT) parental cell line (left lane) and the control-shRNA cells (central two lanes).

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The level of phosphorylation of FAK (Tyr925) correlated with Erc expression in cells tested (Fig. 6a), suggesting that the signals downstream of integrin are upregulated by Erc. Further tests were conducted for the phosphorylation of Akt (Ser473), S6K (Thr389) and rpS6 (Ser235/236) with three Tsc2-related molecules implicated in insulin signaling and mTOR pathway.(38,39) In Erc-restored cells, phosphorylation of Akt (Ser473) and rpS6 (Ser235/236) were found to be more robust while phosphorylation of S6K (Thr389) also was induced although to a lesser extent (Fig. 6a). In other words, the phosphorylation of rpS6 (Ser235/236), catalyzed by S6K as is generally known, was shown to be remarkably weaker than S6K (Thr389) in Erc-deficient cells and induced in Erc-restored cells. The level of phosphorylation of signal transducer and activator of transcription protein 3 (Stat3; Tyr705) is constitutively higher in Erc-restored cells than in Erc-deficient cells. Positive effects of Erc on these phosphorylated events were also verified in Erc-suppressed cells compared with Erc-expressing cells (Fig. 6b).

To investigate the molecular basis for the increase in cell adhesion in the Erc-restored cells, the cells were treated with DMSO (control), 10 μM Akt-I-1/2 (an Akt inhibitor) or 0.1 μM wortmannin (a phosphatidylinositol-3-OH kinase [PI3K] inhibitor), respectively, and then measured the activation of the Akt and the cell adhesion. As shown in Figure 7a, the phosphorylation of Akt (Ser473) was suppressed completely but the phosphorylation of FAK (Tyr925) was not affected in the cells that were treated with an Akt-I-1/2 or wortmannin. The cell adhesion to collagen-coated plates was remarkably decreased in Erc-restored cell (T1-9Ep10) treated by both inhibitors compared with control (DMSO) cells (Fig. 7b). These suggest that the activation of Akt by Erc is dependent on PI3K and the cell adhesion positively regulated by PI3K-Akt pathway because phosphorylation of FAK was not affected by Akt-inhibitor or PI3K-inhibitor treatment.

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Figure 7.  Cell adhesion was decreased by treated with an Akt inhibitor and a PI3K inhibitor. The cells which are the same as Figure 6a, were divided into three groups and treated with DMSO (control), 10 μM Akt-I-1/2 (an Akt inhibitor) or 0.1 μM wortmannin (a PI3K inhibitor), respectively. (a) Western blot analysis was performed with indicated antibodies. The phosphorylation of Akt (Ser473) was suppressed completely with both the inhibitors but the phosphorylation of focal adhesion kinase (FAK; Tyr925) was not affected. (b) The cell adhesion to collagen-coated plates was remarkably decreased in Erc-restored cell treated by both inhibitors compared with control (DMSO) cells. Values are means ± SEM; ***< 0.001, *< 0.01, *< 0.05. Three independent experiments were performed in quadruplicate.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

The Erc gene is highly expressed in renal tumor cells compared to normal renal cells of Tsc2+/− KO mice. Our newly generated Tsc2;Erc double KO mice made it feasible to investigate the function of Erc in carcinogenesis in a mouse tumor model. The results clearly showed that deficiency of Erc decreased the number and size of renal tumors, and reduced cell proliferation and increased apoptosis in Tsc2 KO mice, inhibited cell adhesion to collagen-coated plates, and suppressed tumor formation in nude mice. We also showed that Erc influenced the expression of integrin β1 and phosphorylation of several downstream proteins, such as FAK, Akt, rpS6 and Stat3.

The Tsc2 KO mice and Eker rats, both of which are Tsc2 heterozygous mutants, develop renal tumors through loss of Tsc2 in the WT allele and in multi-steps.(1–5) The human TSC disease is caused by germ-line mutations in either the TSC1 or TSC2 gene, with numerous individual tumors generally arising due to somatic “second-hit” mutations or loss of heterozygosity(24,40) similar to the above animal models. The resulting tumors in humans and in animal models display elevated mTOR signaling, leading to the enhanced phosphorylation of S6K and rpS6.(1–4,41,42) Erc is highly expressed in renal tumor cells of Eker rats and Tsc2 KO mice(6,24) but the state of Erc has not been reported in human TSC disease.

The mTOR pathway has a pivotal function in the coordination of cell metabolism, cell growth and cell proliferation(40–44) but another pathway may be involved in Tsc2 mutant animal models. It has been reported that the administration of rapamycin alone to Tsc2-mutant animal models (KO mice and Eker rats) with established tumors results in tumor regression. This however, is characterized with residual tumor or failure to eradicate microscopic pre-tumorous lesions.(45–47) These results suggested the existence of other pathways involved in the Tsc2-mutant, in addition to the mTOR axis. The Tsc2;Erc double KO mice exploited here allowed us to investigate this putative pathway without the confounding effects of possible drug resistance. Our results strongly supported the existence of another pathway, in addition to the mTOR axis.

Recently, it was reported that hotspots of GPI-anchored proteins and integrin nanoclusters were involved in cell adhesion.(48) This was corroborated by our results that showed the expression of Erc affected the pattern of integrin and phosphorylation of several kinases because Erc is one of the GPI-anchored proteins. The new pathway displaying an Erc-cell adhesion mediated tumor-proliferation function may exist, but no association between Erc and integrin-related signal has been reported. Although Erc expression is associated with the decrease in the amount of mature integrin β1, this phenomenon may be caused by some feedback mechanism from activated cell-adhesion machinery partially regulated by Erc.

Signals from the integrin complex are transmitted through the phosphorylation of FAK.(30–32) Akt is a Tsc2-related molecule implicated in insulin signaling(38,39) known to be activated by cell adhesion-related signaling.(49) Our results showed that the level of phosphorylation of FAK (Tyr925), Akt (Ser473) and rpS6 (Ser235/236) were decreased with Erc-deficient cells (both Erc KO and Erc knock down) while the phosphorylation of S6K (Thr389) was reduced to a lesser extent, suggesting that integrin signaling and downstream proteins were upregulated by Erc.

It is well known that Tsc2-deficiency activates the mTORC1-S6K pathway as well as the downregulation of Akt.(50) However, our results showed the further downregulation of phosphorylation of Akt (Ser473) by Erc-deficiency in Tsc2-deficient cells and upregulation in Erc-restored cells in vitro, suggesting that Erc-deficiency is relevant to the suppression of tumor development in Tsc2+/− mice. The function of Erc might support the receptor signaling through the cell adhesion signaling. Deficiencies of Tsc2 and Erc might be co-operatively involved in tumorigenesis in KO mice.

The important role of Erc in carcinogenesis has attracted a great deal of attention in recent years.(6–22) Several authors reported that overexpression of Erc can accelerate the proliferation and adhesion of cancer cells using cell lines and xenograft models of cancer.(9,10,17–22) Our previous study showed that Erc gene expression silenced by siRNA suppresses tumor growth in the Tsc2 mutant renal carcinoma model.(51) The activation of specific signaling pathways that are important in cancer can lead to an increase in Erc expression. Binding of ovarian cancer antigen CA125/MUC16 to Erc mediates cell adhesion.(17) The overexpression of Erc in pancreatic cancer cells leads to constitutive activation of the transcription factor Stat3, which results in enhanced expression of cyclin E and cyclin E/cyclin-dependent kinase 2 complex formation, as well as increased G1-S transition.(20) Erc inhibits paclitaxel-induced apoptosis through the PI3K pathway.(21) Erc is also differentially regulated by members of the Wnt signal transduction pathway.(22) Combining these reports with our data, multiple possibilities can be considered. First, the pathological high-expression of Erc in specific malignant tumors of humans and in animal models is considered to be the result of carcinogenesis by the mutant Tsc2 or other key gene. The pathological high-expression of Erc plays a prominent role in many signal transduction pathways such as CA125/MUC16, Stat3, PI3K and Wnt, although elucidation of the underlying mechanism remains elusive. Second, since mutant mice in which both copies of the Erc gene were inactivated showed no detectable abnormalities as compared to WT littermates,(23) Erc might have a function specific to carcinogenesis and other pathological conditions. Third, the downregulation of an integrin-related pathway may affect the progression of multi-step carcinogenesis and inhibit the development of large tumors in Tsc2+/−;Erc−/− mice.

In conclusion, we report here that deficiency of Erc affected the integrin-related signal pathway and suppressed the growth of renal tumors in Tsc2 KO mice. An understanding of the signaling pathways and mechanisms of Erc-induced tumor cell adhesion, proliferation and survival may elucidate not only the pathogenesis of renal tumors in Tsc2+/− mice, but also the pathogenesis of other malignant tumors in both animal models and humans. Our experimental system of Tsc;Erc KO mice and cells is useful to unravel the important role of Erc during carcinogenesis, and further analysis of the Erc pathway may help to develop novel anti-cancer therapies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank Nobuo Kamada, Miho Watanabe and Yousuke Kawase (Chugai Research Institute for Medical Science, Inc., Shizuoka, Japan) for mouse production; Youko Hirayama, Hiroaki Mitani, Junko Sakurai (The JFCR-Cancer Institute, Tokyo, Japan), Etsuko Kobayashi, Norihiro Tada, Fumio Kanai, Keiichi Sasahara, Naomi Ohtsuji and Tetsuya Takagaki (Juntendo University School of Medicine, Tokyo, Japan), for technical assistance; Kazunori Kajino, Shuji Momose, Shuji Matsuoka, Xianghua Piao and other members of the Hino laboratory for helpful discussions. This works was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Technology, Sports and Culture, Japan, and Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science, and the Ministry of Health, Labour and Welfare, Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Data S1. Supporting Methods.

Fig. S1. Establishment of expressed in renal carcinoma (Erc) KO mice.

Fig. S2. Amino acid alignment of the expressed in renal carcinoma (Erc)/mesothelin protein.

Fig. S3. Enhanced tumorigenicity of expressed in renal carcinoma (Erc)-restored renal tumor cells transplanted in nude mice.

Fig. S4. Confirmed the characteristics of integrin β1 by treated with lower concentration of tunicamycin.

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CAS_1846_sm_DataS1_FigsS1-S4.pdf542KSupporting info item

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