鉀氯離子共同輸送體在子宮頸癌致癌機轉中角色之研究(3/3)

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行政院國家科學委員會專題研究計畫成果報告

鉀氯離子共同輸送體在子宮頸癌致癌機轉中角色之研究

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計畫類別：個別型計畫

計畫編號： NSC94-2314-B-006-004-

執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日執行單位：國立成功大學醫學系婦產科

計畫主持人：周振陽

計畫參與人員：沈孟儒

報告類型：完整報告

處理方式：本計畫可公開查詢

中華民國 95 年 10 月 30 日

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行政院國家科學委員會專題研究計畫成果報告

鉀氯離子共同輸送體在子宮頸癌致病機轉中角色之研究(3/3) 計畫編號: NSC 92-2314-B-006-124

計畫編號: NSC 93-2314-B-006-021 計畫編號: NSC 94-2314-B-006-004 執行期限: 92 年 8 月 1 日至 93 年 7 月 31 日 執行期限: 93 年 8 月 1 日至 94 年 7 月 31 日 執行期限: 94 年 8 月 1 日至 95 年 7 月 31 日

主持人: 周振陽 執行機構及單位名稱: 成大醫學院婦產學科

一.中文摘要

子宮頸癌是世界上婦女健康的重要問題，但其病態生理致病機轉仍不清楚，我們第一年的研究結果顯示子宮頸癌細胞的生長及侵襲力和其氯鉀離子共同輸送體 (KCC)的活性有很強的相關性。利用細胞 漲大， staurosporine 和 N-ethylmaleimide 來活化KCC 活性的研究顯示從KCC1 去除N 端的117 氨基酸在子宮頸癌細胞會有dominant-negative 失去KCC 功能的表現型。喪失KCC 功能的子宮頸癌細胞其體積調節功能減退，生長速度減緩同時併有細胞週期相關的 Rb 及 cdc2 kinase 功能的變化，這些細胞的侵襲性降低同時併有v 3 ， 6 4 integrin 及 MMP 2 及9 的下降。更重要地，這些KCC 功能減少的子宮頸癌細胞在 SCID 裸鼠上的致癌能力大大地降低本研究顯示抑制 KCC 活性可提供治療或預防子宮頸癌的一種新策略。

第二年本研究主要探討對 IGF-1 接受器(IGF-1 R)的訊息傳遞系統在子宮頸癌形成及轉移所扮演的角色。研究結果發現 IGF-1 可明顯促進子宮頸癌細胞的生長及增加細胞的侵襲性。而上述的作用可以完成被 IGF-1 接受器抗體所抑制。另外，在

71 位早期子宮頸癌病人中，IGF-1 R 的表現和子宮頸癌的旁組織或淋巴結轉移有高度相關，更重要的是 IGF-1 R 的高度表現的病人其預後比起低度表現的病人明顯較差，本研究結果顯示 IGF-1 R 訊息傳遞在未來可作為預防或治療子宮頸癌的一種方法。

在第三年計畫中，因第一型胰島素生長激素（IGF-1）被認為和促進細胞分裂、

轉移和抗細胞凋零的途徑有關連。但是我們對 IGF-1 和 integrin 在子宮頸癌進展中所扮演的角色則是所知很有限。第三年主要探討 IGF-1 感受器（IGF-1R）訊息傳遞的調控機制以及它對子宮頸癌形成的重要性。子宮頸癌細胞的生長及侵襲性受 IGF-1 刺激而增加，正常子宮頸上皮則沒有這種現象。IGF-1R 蛋白在癌細胞株明顯增加，而正常上皮細胞則僅具微量。IGF-1 誘發的生長及侵襲的增加可被 IGF-1R 抗體所抑制，但不受 IgG 或胰島素感受器抗體所抑制。αVβ3integrin 的功能型抗體也可抑制癌細胞的生長及侵襲，但α2、α3、α4、 α6、β1、β4或α2β1的抗體則沒有抑制作用。

進一步的研究發現αVβ3 integrin 抗體可經由改變 SHP2 結合情形來調控 IGF-1R 的磷

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酸化，因而影響 IGF-1 訊息傳遞及其功能。αVβ3也影響 IGF-1R 下游的訊息傳遞因子如 Akt 及 Erk1/2 的活化。經 αVβ3或 IGF-1R 抗體處理後，在 SCID 老鼠接種的子宮頸癌腫瘤明顯受到抑制，其腫瘤組織中的 Akt 及 Erk1/2 的表現量也因抗體作用而明顯降低。綜合言之，IGF-1R 和 αVβ3

integrin 的訊息互動在子宮頸的形成及擴展上扮演了重要的角色。

二. Summary

The first year study： Cervical cancer is a major world health roblem for women, but the pathophysiology of this disease has received scant attention. Here we show that the growth and invasion of cervical cancer cells are strongly linked the expression and activity of the KCl cotransporter (KCC), an important regulator of the ionic and cellular osmotic homeostasis. Functional assays of KCl cotransport activation by osmotic swelling, staurosporine, and N-ethylmaleimide indicate that removal of the N-terminal 117 amino acids from KCC1 produces a dominant-negative loss-of-function phenotype for KCl cotransport in human cervical cancer cells.

The capability for regulatory volume decrease is much attenuated in the loss-of-function KCC mutant cervical cancer cells. The loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle gene products retinoblastoma and cdc2 kinase. Reduced cellular invasiveness is in parallel by reduced expression of v 3 and 6 4 integrins, accompanied by decreased activity of matrix metalloproteinase 2 and 9. Inhibition of tumor growth in SCID mice confirms the crucial role of KCC in promoting cervical cancer growth and invasion. Thus, blockade of KCl cotransport may be a useful therapeutic adjunctive strategy to retard or prevent cervical cancer invasion.

The secondary year study：This study is to test the hypothesis that IGF-1 growth factor receptor signalings may functionally promote cervical cancer development. The results demonstrate that insulin-like growth factor 1 (IGF-1) is a potent stimulator of cervical cancer cell invasion and proliferation. The IGF-1-stimulating effects are completely inhibited by antagonistic antibody against IGF-1 receptor (IGF-1R).

Furthermore, among the 71 patients with early-stage cervical cancer it was found that IGF-1R overexpression was more frequently detected in cervical cancer cells with a strong tendency to invade or metastasize, and was associated significantly with poor patient survival. Thus, blockade of IGF-1R signalings may provide novel strategies for the treatment of invasive phenotypes of cervical cancer.

The third year study ： Insulin-like growth factor 1 (IGF-1) has been implicated in promoting mitogenic, metastatic, and antiapoptotic phenotypes in several types of cancer. But little is known about the signal interaction of IGF-1 and integrin in the regulation of cervical cancer development and progression. This study is to investigate the regulatory mechanism of IGF-1 receptor (IGF-1R) signaling and its importance in cervical cancer formation. The growth and invasiveness of cervical cancer cells (SiHa and CaSki) were dose-dependently stimulated by IGF-1, whereas those of normal cervical epithelial cells were not.

The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against IGF-1R, whereas these cellular functions were unaffected by either IgG or monoclonal antibody to insulin receptor.

Functional-blocking monoclonal antibody against integrinsαvβ3, but not α2,α3,α 4, α 6, β 1, β 4or α 2 β 1,inhibited the

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IGF-1-stimulated invasion and proliferation in cervical cancer cells. α v β 3 integrin modulated IGF-1R phosphorylation by altering the rate of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) recruitment to the activated IGF-1R.

The modulation ofαvβ3 occupancy also affected the activation of IGF-1R downstream-signaling elements, including activation of Akt and extracellular signal-regulated protein kinases 1/2 (Erk1/2).

The treatment of blocking antibody ofαvβ 3 integrin or IGF-1R significantly inhibited tumor growth and caused tumor regression in SCID mice model. Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R and downstream targets of Akt and Erk1/2 were remarkably decreased in SCID mice treated with blocking antibodies of α v β 3 or IGF-1R. Thus, these data suggest that the signal interaction between IGF-1R and α v β 3 integrin plays an important role in promoting the development and progression of cervical cancer.

相關詞：類胰島素生長因子；子宮頸癌 Key words Insulin-link growth factor 1；

cervical cancer；integrin；

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Cervical cancer remains the second most common cancer among women worldwide,accounting for 15% of all female cancers (1).The incidence of invasive cervical cancer varies considerably in different populations,reflecting the influence of variations in environmental factors, Pap smear surveillance and the treatment of pre-invasive lesions.Although much is known about the contribution of oncogenic human papillomavirus (HPV)1 to cervical dysplasia, only a small fraction of those infected eventually develop cancer, indicating that additional factors contribute to the progression to cervical cancer (2, 3).The cellular pathophysiology of this disease remains largely unknown, and very little information is available on the roles of cellular ion transport systems in the neoplastic transformation and progression of cervical epithelial cells.

The KCl cotransporter family (KCC) plays a significant role in the ionic and osmotic homeostasis of many cell types. The Cdna products of four KCC genes have been cloned (4). The activities of KCC1 and KCC4 are osmotically sensitive and involved in volume regulation (5, 6). The neuron-specific KCC2 is critical for the maturation of inhibitory-aminobutyric acid responses in the central nervous system by the control of intracellular Cl–concentration ([Cl–]i) (7). KCC3, initially cloned from vascular endothelial cells (8),may have a physiological significance in cell proliferation (9).

We have demonstrated previously (10)that human cervical carcinogenesis is accompanied by increased expression of mRNA transcripts encoding KCC1, KCC3,and KCC4. owever the lack of specific and potent pharmacological

inhibitors of KCC(except

[(dihydroindenyl)oxy] alkanoic acid(DIOA) at low concentrations) (11) has been a major obstacle to further studies on KCC function

in human cervical carcinogenesis. More direct tests of the physiological functions of KCC and the consequences of loss of KCC activity require knock-out or knockdown experiments. Such experiments will involve either generation of homozygous knock-out animals or transgenic expression of dominant-negative or antisense constructs.

Human cervical cancer cells express more than one KCC gene product. Thus, there is a serious possibility of functional compensation of the knock-out of one gene product by the unaltered or up-regulated expression of cognate genes.

Structure-function studies with KCC1 show that the removal of 117 amino acids from the N-terminal cytoplasmic domain of mouse KCC1 ( N117) confers a dominant-negative phenotype when coexpressed with gene products of human KCC1, KCC3, or mouse KCC4 (12). Using the KCC1 dominant-negative mutants, the present study establishes KCl cotransport as an important modulator of growth and invasiveness of human cervical cancer.

Results

Overexpression of KCC in Human Cervical Cancer—We first examined KCC polypeptide expression in surgical specimens of cervical cancer by immunofluorescent staining with antibody against the N-terminal of human KCC1 (capable of also detecting KCC4(14)). KCC polypeptide was nearly undetectable in the normal cervical epithelial tissues of all surgical specimens examined (n = 30). In contrast, adjacent cervical cancer tissues abundantly expressed KCC polypeptide in cell membrane, as indicated by double staining with Na+/K+ ATPase. The KCC immunofluorescence staining in cancer tissue was absent in the absence of primary antibody and was competed with peptide antigen but not irrelevant peptide.

One of the most significant functions of KCC is to facilitate cell volume regulation(20, 21). As cell swelling is one of the major activating stimuli for KCl

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cotransport (5, 6), we investigated hypotonicity-stimulated KCC activity in various cervical epithelial cell lines representing different stages of cervical carcinogenesis. These included normal primary cervical epithelial cells, HPV-immortalized cells of low (Z172) and high tumor potential (Z183A), and cervical cancer cell lines (SiHa and HT-3). KCl cotransport in normal cervical epithelial cells and in Z172 cells was refractory to cell swelling but could be modestly activated by a hypotonic medium of 200 mosmol/liter. In contrast, KCl cotransport activity in cervical cancer cells (SiHa and HT-3) and in Z183A cells, though not different in isotonic medium, increased sharply as osmolarity was reduced. These results demonstrate a correlation between the osmotic sensitivity of KCl cotransport activity and different stages of cervical carcinogenesis.

Generation of Human Cervical Cancer Cell Lines with Reduced KCl Cotransport Activity—We subsequently developed KCC mutant cervical cancer cell lines stably transfected with the dominant-negative N117 mutant KCC1 cDNA. Further experiments were performed to assess KCC regulation and confirm that N117 transfectants indeed exhibited reduced KCl cotransport. Wild-type cervical cancer cells have KCC activity that is nearly quiescent in isotonic conditions. However high transport rates are observed in response to hypotonic challenge (230 mosmol/liter) and are inhibited by 20 μM DIOA. In contrast, hypotonicity-stimulated KCC activity is completely abolished in N117 mutant cervical cancer cells. Treatment of wild-type cervical cancer cells with 1 μM staurosporine led to a 10-fold stimulation of KCC activity. However, the KCC activity in N117 mutant cervical cancer cells is relatively insensitive to stimulation by staurosporine. Furthermore, whereas 0.5 mM N-ethylmaleimide (NEM) led to a 25-fold stimulation of KCC activity in wild-type cervical cancer cells, KCl cotransport in N117 mutant cervical cancer cells was only slightly increased. [Cl–]i

estimated by 36Cl–equilibrium was 30 ± 2 mM (n = 5) in wild-type cervical cancer cells and 46 ± 3 mM in N117 mutant cells(n

= 5, p < 0.01). Na+/K+ ATPase activity did not differ in the two cell lines (13.4 ± 0.9 versus 14.8 ± 0.7 μmol phosphate/mg protein/h). Mock transfection did not change the regulation of KCC activity, [Cl–]i and Na+/K+ ATPase activity. Taken together, these functional assays indicate that N117 mutant cervical cancer cells exhibit dominant-negative suppression of KCl cotransport activity. Role of KCl Cotransport in Cell Volume Regulation—

Prompted by the observation of substantially increased hypotonicity-stimulated KCl cotransport in cervical cancer cells, we tested the contribution of KCl cotransport activation to enhanced volume regulation of cervical cancer cells by comparison of regulatory volume decrease (RVD) in wild-type and N117 mutant cervical cancer cells. The typical volume response of wild-type cervical cancer cells to hypotonic medium (230 mosmol/liter) can be divided into three phases: (1) an initial, rapid osmotic swelling to reach a peak cell volume; (2) a subsequent, rapid shrinkage in the following 2–3 min; and (3) a later, more gradual decrease of cell volume to reach a plateau value after 10–15 min. Addition of the KCC inhibitor DIOA (20 μM), which should abolish hypotonicity-stimulated KCl cotransport, increased the initial, osmotic swelling, attenuated the shrinkage phase, and inhibited the gradual, slower decrease in cell volume (final volume 122 ± 2% for wild-type cervical cancer cells treated with 20 μM DIOA versus 104 ± 3% without DIOA, p < 0.01). Moreover, the N117 mutant cells showed yet further attenuation of RVD (final volume, 132 ± 1%, p <

0.005).

Remarkably, cervical cancer cells expressing the KCC1 N117 mutant exhibit a lower capacity for volume regulation than do wild-type cervical cancer cells treated with the KCC inhibitor, DIOA. The predominant pathway for RVD of human cervical cancer cells is the opening of the

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volume-sensitive organic osmolyte/anion channel (VSOAC), which leads to Cl–and taurine efflux (15).

Therefore, to further dissect the mechanisms of retarded RVD, the whole-cell mode of the patch clamp technique was used to measure membrane currents. The amplitude and activation rate of VSOAC were significantly decreased in N117 mutant cervical cancer cells.

Consistently, in complementary taurine transport experiments, the swelling-activated taurine efflux rate constant decreased significantly from 0.06 ± 0.002 min–1 (n = 6) in wild-type cervical cancer cells to 0.032 ± 0.001 min–1 (n = 6) in N117 mutant cervical cancer cells (p < 0.01). These results indicate that reduced RVD in N117 mutant cells results from both the near abolition of hypotonicity-activated KCl cotransport and the down-regulation of VSOAC activity.

Suppression of Tumor Growth and Invasion by N117 Mutant—We have reported previously (9) that alteration of [Cl–]I can affect activities of Rb protein and cdc2 kinase, two key cell cycle regulators controlling progression through the restriction point from G1 into S phase and from G2 into M phase, respectively.

Overexpression of the KCC1 mutant N117 elevates [Cl– ]I by decreasing the KCC activity. We therefore hypothesized that the N117 KCC1 overexpression might alter cell proliferation. N117 mutant cervical cancer cells indeed proliferate more slowly than do wild-type cervical cancer cells. This decrease in cell proliferation is accompanied by a decrease in the phosphorylated active form of Rb protein and an increase in the phosphorylated inactive form of cdc2 kinase, suggesting that KCC activity is important for the progression of the cell cycle clock.

The alteration of [Cl–]i has been suggested as an important signal to regulate the activation of integrin in polymorphonuclear leukocytes (22, 23).

Although epithelial cells express a variety of cell adhesion molecules, integrins are the most important extracellular matrix receptors known to play a major role in the

invasion and progression of most types of cancers (24). Abnormal expression of integrins such as v 3, 6 4, 2 1, and 3 1 has been reported in human cervical cancer cells (25–27). To study the

role of integrins in tumor cell invasion, we assayed cell invasion in a Boyden chamber, as an in vitro model for cancer invasion in matrigel (19). Function-blocking monoclonal antibodies against integrins v 3, 6, or 4, but not 2, 3, or 1, potently inhibited the invasion of wild-type cervical cancer SiHa cells, whereas IgG or boiled inactive monoclonal antibodies were without effect.

This indicates that v 3 and 6 4 integrins might play an important role in the invasion of cervical cancer cells. Interestingly, the invasion of wild-type human cervical cancer cells was inhibited in a dose-dependent manner by KCC inhibitor, DIOA. Moreover, invasion is much attenuated in the N117 mutant cervical cancer cells.

Conditioned medium collected from invasion assays was assessed for MMP activity. MMPs comprise a group of enzymes critically involved in the integrin-mediated dissolution of extracellular matrix (24, 28). MMP-2 and MMP-9, highly expressed in wild-type cervical cancer cells, are released as a proenzyme(inactive form) that is proteolytically activatedto the active form.

Compared with wild-type cells, N117 mutant cervical cancer cells showed decreased zymographic activity of MMP-2 (4-fold, p < 0.001) and MMP-9(2-fold, p <

0.01). ELISA determinations confirmed the decreased activities of MMP-2 (4.5-fold, p <

0.001) and MMP-9(2.6-fold, p < 0.01) in the N117 mutant cervical cancer cells.

Because v 3 and 6 4 integrins can play a critical role in cervical cancer invasion and cancer invasion is much attenuated in the N117 mutation, the effect of altered KCC activity on the integrin expression of v 3and 6 4 was examined. Compared with wild-type cells, the N117 mutant cervical cancer cells demonstrated a significant decrease in expression of 6 4 and v 3 integrins in Western blots and in immunofluorescence

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analyses. Taken together, the dominant-negative KCC phenotype is associated with inhibition of cell invasion accompanied by decreased the expression of 6 4 and v 3 integrins and reduced MMP-2 and MMP-9 activities. To avoid clonal variation and selection, we did the same series of functional assays in another four clones of cervical cancer SiHa and HT-3 cells stably transfected with the dominant-negative N117 mutant KCC1 cDNA. Similar results were obtained in the measurement of KCC activity, volume response, electrophysiology, proliferation, invasion ability, and integrin expressions (data not shown). Tumor Growth and Invasion in SCID Mice—Down-regulation of cell proliferation and invasion suggests but does not prove an important role for KCl cotransport in the pathophysiology of human cervical cancer.

To test whether manipulation of KCl cotransport alters tumor growth and invasion in vivo, we inoculated SCID mice subcutaneously with the wild-type or N117 mutant cervical cancer cell lines. Rapid tumor growth was obvious in the groups inoculated with wild-type cervical cancer cells. The N117 mutation significantly reduced the rate of tumor growth and decreased the tumor size 50 days after grafting. The histology of SCID mice xenografts shows the malignant characteristics of wild-type cervical cancer cells, direct invasion into adjacent muscular tissues and blood vessels and formation of infiltrating nests of cells that vary greatly in size and contou. The xenograft specimens resulting from inoculation of N117 mutant cells formed small tumors with intact capsules and without local invasion. In addition, all xenografts resulting from inoculation of N117 mutant cells exhibit an area of central necrosis of varying size.

Moreover, the mitotic figures of wild-type cervical cancer cells are far more numerous than N117 mutant(80 ± 3 versus 14 ± 2 per high powered field(n = 20), p < 0.001).

Consistent with in vitro assay, anti-integrin 4 and v 3 immunofluorescence analysis in

xenograftic tumors resulting from the transplantation of N117 mutant cervical cancer cells showed reduced expression of integrins 4 and v 3. Immunoblots confirmed the presence of N117 KCC1 polypeptide in the small tumors.

B.第二年研究報告 Introduction

Cervical cancer is the second leading cause of cancer death in women worldwide.

There are large differences in incidence rates of invasive cervical cancer among populations, reflecting the influence of environmental factors, screening Pap smears, and the treatment of pre-invasive lesions. A growing body of evidence has accumulated to indicate that oncogenic types of human papillomavirus (HPV) serve as an important factor in the development of the precursors of cervical cancer. The protein products of HPV DNA appear to be interactive with the antioncogenic function of retinoblastoma gene and p53 gene. The pathogenesis of cervical cancer shows multiple stages of progression, from HPV-related cellular growth dysregulation to cervical cancer.

However, only a small fraction of those infected by HPV develop cancer, indicating that other factors contribute to the progression to cervical cancer. Despite intensive studies that have been carried out, the tumor biology of this disease is still largely unknown.

The spread of malignant tumor cells to secondary sites imposes a serious problem in the prognosis and treatment of neoplastic disease. Although prognostic factors such as parametrium invasion and lymph node metastasis affect the outcome of cervical cancer, the variability in progression-free and overall survival among patients with similar clinical and pathological characteristics makes it difficult to predict the outcome reliably. Adhesion receptors of the integrin family promote cell attachment to proteins within the extracellular matrix and potentiate cellular migration and invasion. A correlation has been established between specific integrins and metastatic

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behavior in vivo. Tumor cells can secrete cytokines such as growth factors, which may lead to autocrine stimulation of tumor cell growth and/or motility. Thus, it appears that the interaction of growth factors and adhesion proteins likely contributes to the metastatic and invasive cascades. For example, IGF-1 induces adhesion and migration in human multiple myeloma cells via activation of 1 integrin signalings. In breast cancer MCF-7 cell line, IGF-1-triggered cellular migration and invasion are mediated by _v₅ integrin.

Integrin11and21are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment in response to the stimulation of basic fibroblast growth factor and epidermal growth factor (EGF).

Little is known about how the molecular events of integrin and growth factor receptor regulate the proliferation and invasion of cervical cancer cells. This study is aimed to identify the specific growth factors which are critically involved in cervical cancer cell invasion and proliferation, and test the hypothesis that growth factor receptor signalings may cooperate functionally to promote cancer development and progression.

Results

IGF-1 is a potent stimulator of invasive migration. The invasion assays were performed in cervical cancer SiHa and CaSki cells which have been incubated with different concentrations of various growth factors for 24 hours. As shown in Figure 1A

& 1B, SiHa and CaSki cells migrated in response to the stimulation of IGF-1, EGF and transforming growth factor beta (TGF. Dose-response assays done with both cell types established that 50 ng/ml IGF-1 was an optimal concentration for maximal stimulation of tumor invasion (4.4+0.3-fold

& 4.2+0.4-fold increase compared with DMEM/0.1% FCS alone for SiHa and CaSki cells, respectively), when fibronectin was used aschemoattractin thelowerBoyden’s

chamber. We further compared the invasive migration between normal cervical epithelial cells and cervical cancer cells. Cervical cancer SiHa and CaSki cells presented the better capability of invasion than that of normal epithelial cells in the absence of any stimulation of growth factors (Figure 1C &

1D). Although IGF-1 modestly stimulated the invasion of normal epithelial cells, the invasive capability of SiHa and CaSki cells was markedly enhanced by IGF-1 stimulation when fibronectin or vitronetin was used as chemoattractor (Figure 1C &

1D). This indicates that IGF-1 can differentially enhance the invasive migration between cervical cancer cells and normal cervical epithelial cells. Furthermore, IGF-1-stimulated invasion was completely inhibited by functional-blocking monoclonal antibody against -subunit of IGF-1R in SiHa and CaSki cells (Figure 2A & 2B), whereas the invasion was unaffected by either IgG or monoclonal antibody to IR.

IGF-1 stimulates cervical cancer cell growth. We studied cell proliferation under various culture conditions, to test whether the growth of cervical cancer cells is regulated by IGF-1. As shown in Figure 3A, the growth of cervical cancer cells (SiHa and CaSki) was stimulated dose-dependently by IGF-1, whereas that of normal cervical epithelial cells was not. In addition, the antagonistic monoclonal antibody of IGF-1R, but not IR and IgG, abolished IGF-1-stimulated cancer growth (Figure 3B).

Figure 3C shows the effect of blocking antibodies of IGF-1R on Annexin V binding, as assessed by fluorescence-activated cell sorter analysis. Pictured by the increase in Annexin V binding, IGF-1R blocking antibodies induced an increase in externalization of phosphatidyl serine from the inner leaflet of the plasma membrane, a typical early sign for apoptotic cell death.

This indicates that IGF-1R mediate the survival signalings that inhibit apoptosis.

Patterns of IGF-1, IGF-1R expression in cervical cancer tissues. Studies in cell culture systems have revealed that IGF-1 and IGF-1R signalings are important

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modulators of cervical cancer cell proliferation and invasion. To test the in vivo condition, we examined the expression of IGF-1 and IGF-1R in surgical specimens (n=71) of cervical cancer by immunofluorescent staining. To compare the IGF-1/IGF-1R expression in the different phenotypes of cervical cancer (these surgical specimens), we define the invasive phenotype of cervical cancer as tumors invading the parametrium or metastasizing to pelvic lymph nodes. In contrast, both the parametrium and pelvic lymph nodes are free of tumor in the non-invasive type of cervical cancer. IGF-1 protein was nearly undetectable in normal or noncancerous cervical tissues of all surgical specimens examined (Figure 7B, n=71). In contrast, adjacent cervical cancerous tissues clearly expressed IGF-1 protein. Furthermore, the distribution and intensity of IGF-1 expression were different between non-invasive and invasive phenotypes of cervical cancer (Figure 7B). More than 50%

of cancer tissues in the invasive types of cervical cancer displayed strong IGF-1 stain.

In contrast, the non-invasive phenotype of cervical cancer tissues showed weak IGF-1 stain. The strong IGF-1 stain was also noted in the tumor-invaded parametrium and tumor-metastasized pelvic lymph node (Figure 7B). On the other hand, no IGF-1 stain was found in the tumor-free parametrium and pelvic lymph node. The double stains identify the colocalization of IGF-1 and IGF-1R in cancer tissues (Figure 7C), suggesting autocrine or paracrine stimulation of tumor growth and invasion.

IGF-1R overexpression is a poor prognostic factor. We analyzed the clinical outcome of cervical cancer patients by IGF-1R expression. Table 1 shows the clinicopathological characteristics of cervical cancer patients grouped by the grade of IGF-1R stain. Group 1 includes the grades 1 and 2 of IGF-1R expression which indicates IGF-1R staining intensity and distribution are less than 50% of tumor area.

Group 2 indicates the grades 3 and 4 of IGF-1R expression which shows IGF-1R

staining intensity and distribution are more than 50% of tumor area. Tumor with strong staining of IGF-1R (Group 2) presents significantly higher percentage of parametrium invasion, pelvic lymph node metastasis and larger tumor size (Table 1).

Most importantly, there were significant differences in the distributions of disease-free survival and overall survival according to the presence or absence of IGF-1R overexpression (Figure 9, P<0.005 for both comparison).

Discussion

This study clearly demonstrates that IGF-1 is a potent stimulator of cervical cancer cell invasion and proliferation. The IGF-1-stimulating effects are completely inhibited by antagonistic antibody against IGF-1R, whereas IgG or monoclonal antibody to IR has no effect. This indicates that IGF-1-stimulating effects are specifically mediated through IGF-1R signalings. More importantly, IGF-1 can differentially enhance the invasion and proliferation between cervical cancer cells and normal cervical epithelial cells. Our experiments also show that blockade of IGF1-R occupancy induces apoptosis of tumor cells. This finding is consistent with the proposed antiapoptotic role of IGF1-R signalings (21, 22).

The immunofluorescent studies on surgical specimens further support the notion that IGF-1/IGF1-R pathway contributes to a more aggressive malignant phenotype of cervical cancer. Cervical cancer tissues overexpress both IGF-1 and IGF-1R, as compared with normal or non-cancerous cervical tissue. In addition, IGF-1 and IGF-1R colocalize in cancer tissues, supporting the view that an autocrine or paracrine interaction between IGF1-R and its ligands regulates cell proliferation and invasion. Cervical cancer cells with a strong tendency to invade or metastasize have higher expression of IGF-1/IGF-1R than those with a low ability to do so. Most importantly, IGF-1R overexpression is associated significantly with poor patient survival.

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Take together, this study identifies the IGF-1R/PI3K/Akt pathway contributes to a more aggressive malignant phenotype of cervical cancers (summarized in Figure 11).

Antagonists of IGF-1R signal transduction may provide novel strategies for the treatment of invasive types of cervical cancer.

C.第三年研究報告 Introduction

Cervical cancer is a major woman health problem worldwide (1). A growing body of evidence has accumulated to indicate that oncogenic types of human papillomavirus (HPV) serve as an important factor in the development of the precursors of cervical cancer (2). However, only a small fraction of those infected by HPV develop cancer, indicating that other factors contribute to the progression to cervical cancer (3, 4). Although prognostic factors such as pelvic lymph node metastasis affects the outcome of cervical cancer, the variability in progression-free and overall survival among patients with similar clinical and pathological characteristics makes it difficult to predict the outcome reliably (5).

Specific growth factors significantly enhance the metastatic and invasive properties of cancer cells which pose serious problems to the successful cancer treatment.

The insulin-like growth factor 1 (IGF-1) system has been implicated in promoting mitogenic, metastatic, and antiapoptotic phenotypes in several types of cancer (6, 7).

Each of these properties contributes to IGF-1-mediated maintenance and progression of cancer. Integrins are adhesion receptors that function as cell adhesion and signaling receptors regulating cell death, proliferation, migration, and tissue remodeling (8). One mechanism by which integrin family influence tumor cell progression is through the modulation of growth factor signaling (9). For example,

11and21integrin are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment in response to the stimulation of basic

fibroblast growth factor and epidermal growth factor (EGF) (10). Furthermore,v3

integrin is known to influence growth factor signaling when bound to its ligands, e.g., interaction of v3 integrin with tenascin-c modifies the EGF growth response and results in enhanced EGF receptor activation and downstream signaling (11).

It has been reported that down-regulation of IGF-1 receptor (IGF-1R) by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines (12). But it is unclear about the regulatory mechanism of IGF-1R signalings in cervical cancer cells.

In addition, little is known about the signal interaction of integrin and IGF-1 in the regulation of cervical cancer development and progression. This study is aimed to investigate the regulatory mechanism of IGF-1R signaling and its importance in the formation of cervical cancer. The results show that IGF-1R signaling cooperates with

v3integrin to promote cervical cancer cell proliferation and invasion.

Results

IGF-1 is a potent stimulator of cellular invasion and proliferation. We studied in cell culture systems to test whether IGF-1 is an important modulator of cervical cancer cell invasion and proliferation. Two cervical cancer cell lines (SiHa and CaSki) migrated in response to the stimulation of IGF-1, EGF and transforming growth factor beta.

Dose-response assays done with both cell types established that 50 ng/ml IGF-1 was an optimal concentration for maximal stimulation of tumor invasion, when vitronectin was used as chemoattractor in the lower Boyden’s chamber.In addition, cervical cancer cell lines presented the better capability of invasion than that of normal epithelial cells in the absence of any stimulation of growth factors. Although IGF-1 modestly stimulated the invasive ability of normal cervical epithelial cells, the invasive capability of SiHa and CaSki cervical cancer cells was markedly enhanced by IGF-1 stimulation, when vitronectin or

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fibronectin was used as chemoattractor. In contrast, type IV collagen and laminin did not significantly synergize with IGF-1 to increase the invasiveness of cervical cancel cells.

In proliferation assay, the growth of cervical cancer cells (SiHa and CaSki) was dose-dependently stimulated by IGF-1 (Figure 3A), whereas that of normal cervical epithelial cells was not. The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. This might explain the differential response of normal cervical cells and cancer cells to IGF-1 stimulation.

IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against -subunit of IGF-1R, whereas the invasion and proliferation were unaffected by either IgG or monoclonal antibody to insulin receptor (IR). This indicates that IGF-1-stimulated invasion and proliferation was specific via IGF-1R signalings.

Although epithelial cells express a variety of cell-adhesion molecules, adhesion receptors of integrins are the most important extracellular matrix receptors known to play a major role in the invasion and proliferation of most types of cancers (8, 17).

Expressions of integrin molecules, such as

v3,64, 21, 31 and 41have been reported in human cervical cancer cells (15, 18, 19). We therefore investigated if integrins participate in IGF-1-mediated cellular invasion and proliferation.

Functional-blocking monoclonal antibody against integrins v3, but not 2,3, 4,6,

₁, ₄ or ₂₁, abolished the IGF-1-stimulated invasion and proliferation of cervical cancer SiHa cells and CaSki cells.

This indicates that v3integrin is the key regulator of cervical cancer cell invasiveness across the matrix microenvironment in response to IGF-1 stimulation. Furthermore, Figure 4D shows the effect of blocking antibodies of IGF-1R and _v₃ integrin on Annexin V binding in SiHa cells, as

assessed by fluorescence-activated cell sorter analysis. Pictured by the increase in Annexin V binding, both IGF-1R and _v₃ integrin blocking antibodies induced an increase in externalization of phosphatidyl serine from the inner leaflet of the plasma membrane, a typical early sign for apoptotic cell death. This indicates that IGF-1R and

v3integrin mediate the survival signalings that inhibit apoptosis. Figure 5 further demonstrated that blocking antibodies of IGF-1R and v3 integrin inhibited cervical cell invasiveness and proliferation in a dose-dependent manner.

Mechanisms by which v3 integrin regulates IGF-1R signalings. Blocking

_v₃ integrin occupancy results in attenuation of cellular invasion and proliferation in response to IGF-1 stimulation. The interactions between IGF-1R and integrin receptors were therefore analyzed either by attempting to determine that there was a direct binding of

the two components using

coimmunoprecipitation or showing a change in binding of a known interacting signaling intermediate. We failed to demonstrate a direct physical association between 3

integrin and IGF-1R in cervical cancer SiHa cells. To further define the mechanism by which this interaction occurred, we exposed SiHa cells to IGF-1 or IGF-1 plus blocking antibody of v3 integrin and then immunoprecipitated IGF-1R from cell lysates. We compared the time course of IGF-1R phosphorylation in response to IGF-1 stimulation in the presence or absence of v3 integrin blocking antibody.

Following incubation of cervical cancer SiHa cells with IGF-1, there is a rapid increase in IGF-1R phosphorylation by 5 min. This increase is sustained through 10 min, and is subsequently decreased by 20 min. In contrast, if SiHa cells are preincubated with v3 integrin blocking antibody, there is an equivalent increase in IGF-1R phosphorylation after 5 min. But this phosphorylation is not sustained, in which IGF-1R phosphorylation is significantly decreased after 10 min IGF-1

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treatment.

In smooth muscle cells, the interaction of Src homology 2-containing phosphotyrosine phosphatase (SHP2) with IGF-1R is associated with receptor dephosphorylation (20, 21). Accordingly, we examined whether v3 integrin altered SHP-2 association in cervical cancer cells.

In the presence of blocking antibody ofv3

integrin, there is an increase in the basal association of SHP-2 with IGF-1R before IGF-1 exposure and a remarkable increase in its association with IGF-1R following a 5-min exposure to IGF-1 In contrast, in cells that were not incubated with the blocking antibody, the association of IGF-1R with SHP-2 was not significant until 20 min after IGF-1 stimulation. This implies that the acceleration in the rate of IGF-1R dephosphorylation observed in the presence of blocking antibody of v3 integrin was due to an early recruitment of SHP-2.

We cloned cervical cancer SiHa cells expressing a catalytically inactive SHP-2.

The function of SHP-2 has been reported as part of a positive signaling pathway mediating IGF-1 activation of MAP kinase (22, 23). To analyze whether cervical cancer SiHa cells transfected with SHP-2 mutant are in fact deficient in SHP-2 activity, we measured the phosphorylation level of MAP kinase in response to IGF-1 stimulation.

Figure 7 shows the characterization of SHP-2-deficient clones. SiHa cells transfected with empty vector show normal Erk1/2 activation in response to IGF-1. In contrast, SiHa cells transfected with SHP-2 mutant show a substantial decrease in IGF-1-triggered Erk1/2 phosphorylation. We further studied the time course of IGF-1-stimulated receptor phosphorylation in SiHa cells expressing a catalytically inactive SHP-2. When SiHa cells expressing SHP-2 mutant were exposed to blocking antibody of v3 integrin, it does not result in a decrease of IGF-1R phosphorylation as was seen in cells expressing wild-type SHP-2. This change is not due to the absence of SHP-2 recruitment because cells expressing SHP-2 mutant that were

preincubated with blocking antibody still showed the association of SHP-2 with IGF-1R at 0, 5, and 10 min. This contrasts with the cells expressing only endogenous SHP-2 in which IGF-1R phosphorylation is significantly reduced in the presence of blocking antibody at 10 min, and this reduction is associated with an increase of SHP-2 association with IGF-1R.

To determine whether modulation of

v3 occupancy would affect the IGF-1R-mediated signal transduction, we studied the binding capacity of IGF-1R immediate downstream substrate (IRS) with p85, the regulatory subunit of PI3K. SiHa cells were exposed to IGF-1 alone or the combination of IGF-1 plus antibody to v3

integrin or IGF-1R for 10 min. Antibody was added 30 min before the addition of IGF-1. After a 10-min incubation at 37°C, the cells were lysed and immunoprecipitated by IRS-1 or IRS-2, and then the immunoprecipitated proteins were separated by SDS/PAGE and immunoblotted for p85 subunit of PI3K. The addition of antibody to

v3 integrin or IGF-1R almost abolished the binding of p85 with IRS-2. In contrast, no association between p85 and IRS-1 was noted.

One of the important downstream signaling molecules in PI3K pathway is Akt.

As shown in Figure 9B, IGF-1 treatment resulted in Akt phosphorylation, which was abolished by_v₃ integrin antibody and two structurally different PI3K inhibitors (LY294002 or wortmanin). Similarly, _v₃ integrin occupancy also abolished IGF-1R-mediated signal transduction of Erk1/2 activation.

Inhibition of cervical cancer growth and development in vivo. To test whether manipulation of IGF-1R signalings alters tumor growth and invasion in vivo, we inoculated SCID mice subcutaneously with cervical cancer SiHa cells. Rapid tumor growth was obvious in the control groups. In contrast, the treatment of blocking antibody of v3 integrin or IGF-1R inhibited tumor growth in a dose-dependent manner.

Immunoblots of tumor tissues confirmed

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that the phosphorylation of IGF-1R decreased as a function of time in SCID mice treated with blocking antibodies of

v3 or IGF-1R. In addition, IGF-1R downstream targets of Akt and Erk1/2 were remarkably decreased in the groups with antibody treatment.

We also did the experiments to study whether the blocking antibodies have a role in tumor regression. When tumor xenografts had reached a volume of about 400 mm³, the mice were randomized by tumor volume into three groups of six animals each. The three groups were treated every three days with control IgG, blocking antibody of v3

integrin or IGF-1R, respectively. As shown in Figure 12, the treatment of blocking antibodies significantly caused tumor regression which could sustain about 10-12 days after antibody withdrawing.

Discussion

Here we show that IGF-1R signaling is important for cervical cancer cell invasiveness and proliferation. This conclusion is supported by the following findings: (i) IGF-1R proteins are abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. (ii) IGF-1 is a potent stimulator of cellular invasion and proliferation in cell culture systems. (iii) The IGF-1-stimulated effects are completely inhibited by antagonistic antibody against IGF-1R, whereas IgG or monoclonal antibody to IR has no effect. (iv) The manipulation of IGF-1R signalings by blocking antibody of IGF-1R inhibits tumor growth and causes tumor regression in SCID mice model.

This study identifies an important role of α v β 3 integrin which functions as a signal modulation of IGF-1R biochemical outputs in cervical malignancy. Inhibition of ligand occupancy of α v β 3 integrin by blocking antibody results in attenuation of cervical cancer cell response to IGF-1 stimulation, such as receptor phosphorylation, subsequent signal transduction and cellular function. The

antagonistic antibody used here is a purified monoclonal blocking α v β 3 integrin antibody (clone LM609, Chemicon, CA) and IGF-1R antibody (catalog number MAB1122, Chemicon, CA). Several studies have shown that these antibodies could inhibitαvβ3 integrin dependent (24, 25) or IGF-1R dependent function (26, 27), respectively. Accordingly, there is a specific linkage between the activation ofα v β 3 integrin and IGF-1R-mediated signal transduction pathways in cervical cancer cells. The molecular mechanism by whichα v β 3 actually functions to alter IGF-1 receptor phosphorylation could be mediated through multiple types of interactions.

Because these two proteins do not appear to coimmunoprecipitate, it is likely that blocking occupancy ofαvβ3 integrin by antibody directly affects an intermediary protein that binds to IGF-1R. The dephosphorylation of IGF-1R caused by blockade of α v β 3 occupancy was accompanied by increasing SHP2 recruitment to IGF-1R. This is consistent with the findings in porcine aortic smooth muscle cells which showαvβ3 integrin regulates IGF-1R phosphorylation by altering the rate of SHP-2 recruitment to the activated IGF-1R (20, 21, 28). Therefore, SHP-2 is a candidate protein responsible for the cross talk between the activation ofαv β3 integrin and IGF-1R-mediated signal transduction pathways. Further experiments on cervical cancer cells expressing a catalytically inactive SHP-2 confirm that the acceleration in the rate of IGF-1R dephosphorylation observed in the presence of αvβ3 integrin antibody was due to an early recruitment of SHP-2.

It has been reported that blocking the adhesion receptor of integrin family would inhibit IGF-1 signalings. For example, inhibiting integrin function of  α 5 β 1 blocks the IGF-1 stimulation of cell motility in a metastatic breast cancer cell line, MDA-231BO (29). In smooth muscle cells, blocking ligand occupancy ofαvβ3 with the distintegrin echistatin reduces

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IGF-1-stimulated receptor phosphorylation, and it also inhibits cellular migration and DNA synthesis responses to IGF-1 (30).

Blockingβ1 integrin inhibits IGF-1-induced cell adhesion to fibronectin in human multiple myeloma cells (31). Here we identified the specific integrin, α v β 3, which is critically involved in IGF-1-mediated growth and invasion of cervical cancer cells. Although α 5 β 1 integrin is expressed in cervical cancer cells (our unpublished observation), its role in IGF-1-induced signalings is not clear and needs to be explored in the future.

Take together, this study identifies an unexpected role for α v β 3 integrin in cervical cancer progression and development as a functional amplification of biochemical outputs rather than a mechanical adhesion. IGF-1 stimulates the invasion and proliferation in human cervical cells via the cooperation ofαvβ3 integrin.

Therefore, blockade ofαvβ3 integrin and IGF-1R signal transduction may provide a novel strategy for the treatment of cervical cancer.

四.References

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(1992) Natural history of cervical human papillomavirus lesions does not substantiate the biologic relevance of the bethesda system. Obstet. Gynecol., 79, 675-682.

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(1999) Integrin signaling. Science, 285, 1028-1032.

9. Varner, J.A. and Cheresh, D.A. (1996) Integrins and cancer. Curr. Opin. Cell Biol., 8, 724-730.

10. Yang, C., Zeisberg, M., Lively, J.C., Nyberg, P., Afdhal, N. and Kalluri, R. (2003) Integrin  1 1 and  2 1 are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment.

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11. Swindle, C.S., Tran, K.T., Johnson, T.D., Banerjee, P., Mayes, A.M., Griffith, L.

and Wells, A. (2001) Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor. J.

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(2000) Down-regulation of the insulin-like growth factor I receptor by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines. Cancer Res., 60, 760-765.

13. Shen, M.R., Wu, S.N. and Chou, C.Y. (1995) Volume-sensitive chloride channels associated with human cervical carcinogenesis. Cancer Res., 55, 6077-6083.

14. Maile, L.A. and Clemmons, D.R.

(2002) The  V 3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor.

Endocrinology, 143, 4259-4264.

15. Shen, M.R., Chou, C.Y., Hsu, K.F., Hsu, Y.M., Chiu, W.T., Tang, M.J., Alper, S.L. and Ellory, J.C. (2003) KCl

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cortransporter is an important modulator of human cervical cancer growth and invasion.

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16. Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M. and McEwan, R.N. (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res., 47, 3239-3245.

17. Juliano, R.L. and Varner, J.A. (1993) Adhesion molecules in cancer: the role of integrins. Curr. Opin. Cell Biol., 5, 812-818.

18. Aplin, J.D., Dawson, S. and Seif, M.W. (1996) Abnormal expression of integrin  6 4 in cervical intraepithelial neoplasia. Br. J. Cancer, 74, 240-245.

19. Jeffers, M.D., Paxton, J., Bolger, B., Richmond, J.A., Kennedy, J.H. and McNicol, A.M. (1997) E-cadherin and integrin cell adhesion molecule expression in invasive and in situ carcinoma of the cervix. Gynecol.

Oncol., 64, 481-486.

(2002) The  V 3 integrin regulates insulin-like growth factor I (IGF-I) receptor phosphorylation by altering the rate of recruitment of the src-homology 2-containing phosphotyrosine phosphatase-2 to the activated IGF-I receptor.

Endocrinology, 143, 4259-4264.

21. Ling, Y., Maile, L.A. and Clemmons, D.R. (2003) Tyrosine phosphorylation of the  -subunit of3 thevß3 integrin isrequired formembrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor. Mol.

Endocrinol., 17, 1824-1833.

22. Zhao, Z., Tan, Z., Wright, J.H., Diltz, C.D., Shen, S.H., Krebs, E.G. and Fischer, E.H. (1995) Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

J Biol. Chem., 270, 11765-11769.

23. Manes, S., Mira, E., Gomez-Mouton, C., Zhao, Z.J., Lacalle, R.A.

and Martinez-A C. (1999) Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility.

Mol. Cell. Biol., 19, 3125-3135.

24. Doerr, M.E. and Jones, J.I. (1996) The roles of integrins and extracellular matrix proteins in the insulin-like growth factor-I stimulated chemotaxis of human breast cancer cells. J. Biol. Chem., 271, 2443–2447.

25. Jones, J.I., Prevette, T., Gockerman, A. and Clemmons, D.R. (1996) Ligand occupancy of the  V 3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor I.

Proc. Natl. Acad. Sci., 93, 2482-2487.

26. Soos, M,A., Field, C.E., Lammers, R., Ullrich, A., Zhang, B., Roth, R.A., Andersen, A.S., Kjeldsen, T. and Siddle, K.

(1992) A panel of monoclonal antibodies for the type I insulin-like growth factor receptor.

Epitope mapping, effects on ligand binding, and biological activity. J. Biol. Chem., 267, 12955-12963.

27. Schumacher, R., Soos, M.A., Schlessinger, J., Brandenburg, D., Siddle, K.

and Ullrich, A. (1993) Signaling-competent receptor chimeras allow mapping of major insulin receptor binding domain determinants. J. Biol. Chem., 1268, 1087-1094.

(2002) Regulation of insulin-like growth factor I receptor dephosphorylation by SHPS-1 and the tyrosine phosphatase SHP-2.

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29. Zhang, X. and Yee, D. (2002) Insulin-like growth factor binding prptein-1 (IGFBP-1) inhibits breast cancer cell motility. Cancer Res., 62, 4369-4375.

30. Zheng, B. and Clemmons, D.R.

(1998) Blocking ligand occupancy of the V 3 i

  ntegrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells. Proc. Natl. Acad. Sci., 95, 11217-11222.

31. Tai, Y.T., Podar, K., Catley, L., Tseng, Y.H., Akiyama, M., Shringarpure, R., Burger, R., Hideshima, T., Chauhan, D., Mitsiades, N., Richardson, P., Munshi, N.C., Kahn, C.R., Mitsiades, C. and Anderson, K.C. (2003) Insulin-like growth factor-1 induces adhesion and migration in human

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multiple myeloma cells via activation of

 -integrin1 and phosphatidylinositol 3'-kinase/AKT signaling. Cancer Res., 63, 5850-5858.

五、計劃導致的著作

1. Shen MR, Chou CY, Hsu KF, hsu YM, Chiu WT,Tang MJ, Alper SL, Ellory JC.

KCl cotransport isan important modulator of the human cervicalcancer growth and invasion. J Biol Chem (USA)2003:278;

39941-39950（附錄一）

2.Shen MR, YM Hsu, KF Hsu, YF Chen, MJ Tang, CY Chou. Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation which is modulated byαvβ3 integrin signaling. Carcinogenesis 27, 962-971, 2006（附錄二）

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Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by avb3 integrin signaling

Meng-Ru Shen^1,2,5, Yueh-Mei Hsu³, Keng-Fu Hsu², Yih-Fung Chen³, Ming-Jer Tang^4,5and

Cheng-Yang Chou^2,5,

1Department of Pharmacology,²Department of Obstetrics and Gynecology,

3Institute of Basic Medical Sciences,⁴Department of Physiology, College of Medicine and⁵Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 704, Taiwan

To whom correspondence should be addressed at: Dr Cheng-Yang Chou, Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 704, Taiwan Tel:+886 6 2353535 ext. 5608; Fax:+886 6 2766185;

Email: [email protected]

Insulin-like growth factor 1 (IGF-1) has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer. But little is known about the signal interaction of IGF-1 and integrin in the regulation of cervical cancer development and progression.

This study is to investigate the regulatory mechanism of IGF-1 receptor (IGF-1R) signaling and its importance in cervical cancer formation. The growth and invasiveness of cervical cancer cells (SiHa and CaSki) were dose- dependently stimulated by IGF-1, whereas those of normal cervical epithelial cells were not. The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. IGF-1- stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against IGF- 1R, whereas these cellular functions were unaffected by either IgG or monoclonal antibody to insulin receptor.

Functional-blocking monoclonal antibody against integrinsavb3, but nota2,a3,a4,a6,b1,b4ora2b1, inhibited the IGF-1-stimulated invasion and proliferation in cervical cancer cells. avb3 integrin modulated IGF-1R phosphorylation by altering the rate of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) recruitment to the activated IGF-1R. The modulation of avb3 occupancy also affected the activation of IGF-1R downstream-signaling elements, including activation of Akt and extracellular signal-regulated protein kinases 1/2 (Erk1/2). The treatment of blocking antibody ofavb3

integrin or IGF-1R significantly inhibited tumor growth and caused tumor regression in SCID mice model.

Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R and downstream targets of Akt and Erk1/2 were remarkably decreased in SCID mice treated with blocking antibodies of avb3 or IGF-1R. Thus, these data suggest that the signal interaction

between IGF-1R anda_vb₃integrin plays an important role in promoting the development and progression of cervical cancer.

Introduction

Cervical cancer is a major woman health problem worldwide (1). A growing body of evidence has accumulated to indicate that oncogenic types of human papillomavirus (HPV) serve as an important factor in the development of the precursors of cervical cancer (2). However, only a small fraction of those infected by HPV develop cancer, indicating that other factors contribute to the progression to cervical cancer (3,4). Although prognostic factors such as pelvic lymph node metastasis affects the outcome of cervical cancer, the variability in progression- free and overall survival among patients with similar clinical and pathological characteristics makes it difficult to predict the outcome reliably (5).

Specific growth factors significantly enhance the metastatic and invasive properties of cancer cells, which pose serious problems to successful cancer treatment. The insulin-like growth factor 1 (IGF-1) system has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer (6,7). Each of these properties contributes to IGF-1-mediated maintenance and progression of cancer. Integrins are adhesion receptors that function as cell adhesion and signaling receptors regulating cell death, proliferation, migration and tissue remodeling (8). One mechanism by which integrin family influence tumor cell progression is through the modulation of growth factor signaling (9). For example, a1b1 and a2b1 integrin are the key regulators of hepatocarcinoma cell invasion across the fibrotic matrix microenvironment in response to the stimulation of basic fibroblast growth factor and epidermal growth factor (EGF) (10).

Furthermore,avb3integrin is known to influence growth factor signaling when bound to its ligands, that is, interaction ofavb3

integrin with tenascin-c modifies the EGF growth response and results in enhanced EGF receptor activation and downstream signaling (11).

It has been reported that downregulation of IGF-1 receptor (IGF-1R) by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines (12). But the regulatory mechanism of IGF-1R signalings in cervical cancer cells is not clear. In addition, little is known about the signal interaction of integrin and IGF-1 in the regulation of cervical cancer development and progression. This study is aimed at investigating the regulatory mechanism of IGF-1R signaling and its importance in the formation of cervical cancer. The results show that IGF-1R signaling cooperates withavb3integrin to promote cervical cancer cell proliferation and invasion.

Abbreviations:EGF, epidermal growth factor; Erk1/2, extracellular signal- regulated protein kinases 1/2; IGF-1R, insulin-like growth factor 1 receptor;

IRS, insulin receptor substrate; KSFM, keratinocyte serum-free medium;

MAP, mitogen-activated protein; PI3K, phosphatidylinositol-3 kinase; SHP-2, Carcinogenesisvol.27 no.5 pp.962–971, 2006

doi:10.1093/carcin/bgi336

Advance Access publication January 7, 2006