Targeting ILK and β4 integrin abrogates the invasive potential of ovarian cancer

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Abstract

Integrins and integrin-linked kinase (ILK) are essential to cancerous invasion because they mediate physical interactions with the extracellular matrix, and regulate oncogenic signaling pathways. The purpose of our study is to determine whether deletion of β1 and β4 integrin and ILK, alone or in combination, has antitumoral effects in ovarian cancer. Expression of β1 and β4 integrin and ILK was analyzed by immunohistochemistry in 196 ovarian cancer tissue samples. We assessed the effects of depleting these molecules with shRNAs in ovarian cancer cells by Western blot, conventional RT-PCR, cell proliferation, migration, invasion, and in vitro Rac1 activity assays, and in vivo xenograft formation assays. Overexpression of β4 integrin and ILK in human ovarian cancer specimens was found to correlate with tumor aggressiveness. Depletion of these targets efficiently suppresses ovarian cancer cell proliferation, migration, and invasion in vitro and xenograft tumor formation in vivo. We also demonstrated that single depletion of ILK or combination depletion of β4 integrin/ILK inhibits phosphorylation of downstream signaling targets, p-Ser 473 Akt and p-Thr202/Tyr204 Erk1/2, and activation of Rac1, as well as reduce expression of MMP-2 and MMP-9 and increase expression of caspase-3 in vitro. In conclusion, targeting β4 integrin combined with ILK can instigate the latent tumorigenic potential and abrogate the invasive potential in ovarian cancer.

Highlights

► The potential of targeting ILK and integrins for highly aggressive ovarian cancer. ► Unanticipated synergistic effect for the combination of ILK/β4 integrin. ► Combination of ILK/β4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade. ► Targeting of β4 integrin/ILK had potent inhibitory effects in ovarian cancer.

Introduction

Integrin signaling is regulated by complex interactions with a number of cytosolic proteins, including integrin-linked kinase (ILK). ILK is a ubiquitously expressed protein serine/threonine kinase that was initially discovered through its interactions with the β1 and β3 integrin subunits [1], [2]. Although ILK initially named as a kinase, ILK acts as a central component of ILK–PINCH–Parvin complex at ECM adhesions mediating interactions with a large number of proteins via multiple sites including its pseudoactive site [3], [4], [5], [6].

Now many studies have reported that ILK plays a role as an adaptor and signaling protein in various aspects of the oncogenic process through direct and indirect mechanisms during tumor progression [7], [8]. Recent reports showed that aberrant ILK mediated signaling, due to overexpression or constitutive activation of the protein, leads to pathological alterations that ultimately result in malignant progression in a range of cancers [9], [10].

ILK expression is increased in ovarian epithelial cancer relative to benign tumors and normal ovarian epithelium, correlates with increased tumor grade, and is stimulated by soluble factors in peritoneal tumor fluid through the activation of the downstream protein kinase B/Akt pathway [11], [12]. In addition, a recent study showed that ILK directly mediated actin cytoskeletal rearrangements and cell migration and invasion through the concerted actions of phosphoinositide 3-kinase (PI3K)/Akt/Rac1 [13].

Meanwhile, β1 integrin expressed on metastatic ovarian cancer cells affects adhesion to the mesothelium. It has been proposed that ovarian cancer metastasis is regulated by β1 integrin binding to the fibronectin secreted by mesothelial cells [14]. More recent studies also demonstrated that high levels of α4β1 and or αvβ3 integrins were closely correlated with increased peritoneal metastasis and tumor proliferation in ovarian cancer, respectively [15], [16].

Furthermore, the expression of α6 and β4 integrin subunits in serous ovarian carcinoma correlates with expression of the basement membrane protein laminin. In most solid ovarian tumors, expression of laminin is patchy or absent in the putative basement membrane zone surrounding the nest of epithelial tumor cells. In addition, neither laminin or the α6 or β4 integrin subunits are present on the surface of ovarian carcinoma cells isolated from the ascites fluid of most patients, regardless of whether they are present in the patient’s solid tumor. Thus, it is possible that ovarian carcinoma epithelial cells may be released from the basement membrane of the ovary due to their deficit of α6 and β4 integrin subunits [17]. However, the role of α6 or β4 integrin subunits in modulating the phenotypic behavior of ovarian carcinoma cells has not been thoroughly investigated and is poorly understood.

Accordingly, we hypothesized that overexpression of ILK and integrin β subunits in highly oncogenic cancer cells is related to ovarian cancer progression. We determined if targeting these molecules has antitumor effects for ovarian cancer. Furthermore, we addressed the question of whether there is a complementary and synergistic advantage when these molecules were targeted alone or in combination in highly oncogenic human ovarian cancer cells.

Section snippets

Preparation of ovarian cancer patient samples for immunohistochemistry

We examined medical records and archival slides from the collection of ovarian serous adenocarcinoma of the Gynecologic Oncology Files of Yonsei University College of Medicine in Korea. One hundred ninety-six samples of ovarian serous carcinomas were isolated between 1990 and 2003 and used to create tissue microarrays with 2-mm pores in 3.8-cm × 2.2-cm × 0.5-cm frames. The detailed immunohistochemistry procedures and antibody information are provided in Supplementary materials and methods.

ILK and β4 integrin are expressed in highly oncogenic human ovarian cancer cells

We evaluated the expression level of β1, β3, and β4 integrin subunits and ILK in 3 epithelial ovarian cancer cell lines of different histopathological types by Western blot analysis. We showed that β1 integrin was highly expressed in all cell lines, whereas β3 and β4 integrins and ILK were differentially expressed (Fig. 1A-a, left panel). Integrin subunit β3 was highly expressed in TOV-112D cell. In particular, ILK and β4 integrin were highly expressed in the highly oncogenic and invasive

Discussion

We investigated the potential of targeting integrins and ILK as combination therapy for highly aggressive ovarian cancer. Our data showed that targeting ILK alone and ILK in combination with β4 integrin effectively inhibited the PI3K/Akt/Rac1 cascade in invasive human ovarian cancer cell line SK-OV-3 by blocking the activation of Akt and Rac1 in in vitro and in vivo assays. In particular, our data discloses for the first time an unanticipated role and a synergistic effect for the combination of

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2011-0009444; CNH) and by Mid-career Researcher Program through NRF grant funded by the MEST (No. 2012-029435; CNH).

References (17)

  • K. Lessan et al.

    CD44 and beta1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells

    Am. J. Pathol.

    (1999)
  • C.N. Landen et al.

    Tumor-selective response to antibody-mediated targeting of alphavbeta3 integrin in ovarian cancer

    Neoplasia

    (2008)
  • G.E. Hannigan et al.

    Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase

    Nature

    (1996)
  • S. Dedhar et al.

    Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signaling

    Trends Cell Biol.

    (1999)
  • Y. Tu et al.

    The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells

    Mol. Cell. Biol.

    (1999)
  • F. Li et al.

    Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

    Mol. Cell. Biol.

    (1999)
  • B.P. Chiswell et al.

    The structural basis of integrin-linked kinase-PINCH interactions

    Proc. Natl. Acad. Sci. USA

    (2008)
  • S.A. Wickström et al.

    The ILK/PINCH/parvin complex: the kinase is dead long live the pseudokinase!

    EMBO J.

    (2010)
There are more references available in the full text version of this article.

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