**2. Integrins in ovarian cancer and the significance of ascites**

## **2.1 Integrins and integrin-associated survival pathways in ovarian cancer**

The potential role of integrins in critical processes leading to ovarian cancer progression, including the detachment of cancer cells from primary sites, spheroid formation, migration, adhesion to secondary sites, and invasion has been reported by multiple groups [17–28]. The clustering of collagen-binding integrins α2β1 and α3β1 is associated with increased expression and activity of matrix metalloproteinase-9 (MMP-9). An increase in activated MMP-9 is associated with the shedding of E-cadherin, a transmembrane glycoprotein that regulates cell-to-cell adhesion, and increased epithelial-mesenchymal transition (EMT), changes that are indicative of an invasive and metastatic phenotype in ovarian cancer cells [17–20]. The αvβ6 integrin has also been associated with protease secretion and ECM degradation in ovarian cancer cell lines, both of which are indicators of invasive potential [21–23]. Collagenbinding integrins, including heterodimer α4β1, have also been implicated in ovarian cancer migration by Slack-Davis *et al.*, who showed that transmigration of SKOV3 cells through a mesothelial monolayer model decreased significantly upon blocking of α4 integrin, β1 integrin, or vascular cell adhesion protein-1 (VCAM-1). VCAM-1 is a glycoprotein that is predominantly expressed on endothelial cells, but, under high levels of inflammation and in chronic pathological conditions, is also expressed on other cell types, including macrophages and cancer cells [29]. The aforementioned findings by Slack-Davis *et al.* suggest that the VCAM-1-α4β1 integrin interaction is involved in ovarian cancer cell metastasis and invasion through the mesothelium [24]. In addition to migration and invasive potential, studies have found that α5 and β1 integrins are critical for ovarian cancer cell spheroid formation as well as their adhesion to different ligands including fibronectin, laminin and collagen IV, further implicating integrins in ovarian cancer progression [25–28].

In the context of integrins, disease progression, and drug resistance in ovarian cancer, cell signaling pathways, including PI3K/Akt, Ras/Raf/MEK/ERK, Wnt, YAP/ TAZ, as well as crosstalk between integrins and the epidermal growth factor receptor (EGFR), have been most commonly investigated (**Figure 2**) [30–35]. A key player in the activation of the aforementioned pathways is focal adhesion kinase (FAK), a tyrosine kinase that localizes to focal adhesions [34, 36–40]. The overexpression of FAK is frequently associated with advanced-stage ovarian cancer and with increased invasiveness [41], thus FAK inhibition has been investigated as a treatment approach for ovarian cancer [42, 43]. The following subsections describe the current state of the literature on integrin-mediated activation of key molecules and survival pathways that contribute to ovarian cancer progression.

## *2.1.1 PI3K/Akt pathway*

The PI3K/Akt pathway transduces signals from the cell membrane to the cytoplasm and mediates fundamental cellular functions including proliferation and survival [44, 45]. Upon activation by a growth factor, receptor tyrosine kinases (RTKs)

### **Figure 2.**

*Integrin activation can influence cell survival pathways: (1) the PI3K/Akt survival pathway involves the recruitment of FAK to focal adhesions. FAK can propagate the PI3K/Akt pathway, either directly or through Src kinase. (2) Shc phosphorylation by both Src and FAK, which initiates the Shc-Grb2-Sos-Ras cascade may lead to ERK phosphorylation. (3) FAK activation by integrins may also lead to the activation of Wnt/β-catenin pathway. Crosstalk between EGFR and integrins can potentiate signaling and cooperatively stimulate intracellular pathways that contribute to cell survival and drug resistance.*

activate PI3K and trigger its conversion from phosphatidylinositol-4, 5-bisphosphate (PIP2) to phosphatidylinositol-3, 4, 5-triphosphate (PIP3). The serine/threonine kinase Akt interacts with PIP3, which causes its translocation to the plasma inner membrane, where it is phosphorylated by phosphatidylinositide-dependent kinase 1 (PDK1) and PDK2, known as Ser473-phosphorylated Akt kinase. The phosphorylated and activated Akt may interact with substrates that regulate cell growth and survival, including mTOR, Glycogen synthase kinase-3 (GSK3), Bad, and caspase-9. The PI3K/ Akt pathway can also be activated by other cell surface receptors such as cytokine receptors and integrins. A recent study Zheng *et al.*, found that α2β1 overexpressing (α2β<sup>1</sup> + ) ovarian cancer cells, and ovarian cancer patient tissue samples that were resistant to microtubule-directed chemotherapeutic drugs, including paclitaxel and vincristine, had enhanced PI3K and Akt phosphorylation, as well as Akt translocation into the nucleus [30]. This suggests that α2β1 integrins activate the PI3K/AKT pathway to promote resistance to microtubule-directed chemotherapeutic drugs.

Integrin-mediated activation of PI3K/Akt survival pathway involves the recruitment of FAK to the adhesion complex [36]. FAK interacts with the cytoplasmic tail of β-subunits on integrins and forms a dual kinase complex with c-Src. FAK can activate the PI3K/Akt pathway, either directly or through Src kinase. The relationship between FAK signaling and PI3K/Akt pathway-mediated resistance to taxane-based therapy has been demonstrated in a study Kang *et al*. The authors found that VS-6063, a FAK inhibitor, synergized with paclitaxel in HeyA8-MDR cells and showed an additive inhibitory effect with paclitaxel in the taxane resistant cell lines SKOV3-TR and SKOV3ip1 [42]. Decreased tumor weight was also reported in the same study in mouse models of these cell lines after treatment with paclitaxel and VS-6063 compared to paclitaxel alone. Others have also explored the effectiveness of VS-6063 in ovarian cancer growth inhibition [43]. Xu *et al.* screened combinations of VS-6063 with 30
