In the last decades, drug resistance to several tumor targeted therapies has been largely demonstrated, despite the subtle mechanisms that sustain tumor cells between initial response and disease progression are not completely understood.1 An interesting strategy to overcome drug resistance relies on the development of multi-target therapy with the aim to inhibit different targets involved in the tumor genesis and development and, most importantly, to address their intertwined crosstalk.2
The crosstalk between certain integrin receptors and Growth Factor Receptors (GFRs) plays a pivotal role in the development of drug resistance in solid tumors.3 GFRs are membrane-bound glycoproteins and belong to the large family of receptors that have tyrosine kinases activity (RTK).
The binding of their natural ligands triggers multiple intracellular cascades (e.g. PI3K, MAPKs, ERK,
…), making them key regulators of cell proliferation, survival and growth. These receptors are frequently overexpressed or amplified in a wide variety of solid tumors, and mutations affecting their expression or activity confer a growth advantage to the cells and predispose them to malignant transformations.3
Hence, tyrosine kinase inhibitors (TKIs), either monoclonal antibodies or small molecules, have been developed to interrupt several GFR-related pathways. For instance, sunitinib (1, Figure 1) is an alkylidene 2-oxindole derivative with antiangiogenic activity, acting as a highly effective multitarget tyrosine kinase inhibitor (mainly against VEGFR2, PDGFRβ, c-Kit, and Flt-3) and it is indicated as a first-line treatment against metastatic renal cell carcinoma or neuroendocrine tumors.4
Figure 1. Structure of the TKIs sunitinib (1) and nintedanib (2).
Another structurally similar, potent TKI is nintedanib (2, Figure 1), which targets three major pro-angiogenic and pro-fibrotic pathways mediated by the VEGFRs, FGFRs and PDGFRs, Src and Flt-3 kinases. It is clinically approved for the treatment of Idiopathic Pulmonary Fibrosis (IPF) and recent clinical evidences have shown nintedanib having significant efficacy in the treatment of non-small cell lung cancer (NSCLC) and ovarian cancer.5 Both these small molecules are multi-kinase inhibitors, a seemingly desirable characteristic for drugs in the treatment of both cancer and fibrosis-related diseases; in fact, it has been demonstrated that the use of multi-kinases inhibitors is beneficial in overcoming tumor resistance and increasing therapeutic success as compared to selective kinase inhibitors.5
Interestingly, recent studies have highlighted the impact of integrin-mediated signalling in TKI-cancer therapy resistance,3,6 but how integrins regulate response to this class of drugs remains
113 debated, despite the role of these receptors (in particular the V integrins) and their crosstalk with GFRs have been largely described.3,6-8 The most likely hypothesis is the mutual cooperation between the two distinct receptor systems in which (i) integrins induce ligand-independent activation of GFRs and, on the other hand, (ii) growth factors can induce adhesion molecules to propagate adhesion-independent signals.9 In fact, the signals triggered by either GFRs or integrins might follow parallel and often superimposable pathways, that converge on common downstream effectors (Figure 2a) leading to a therapy-resistant tumor.
Figure 2. Scheme of the crosstalk between integrins and GFRs: (a) and (b) mechanism of αV3-mediated resistance in tumors treated with EGFR-TKI. Adapted from Refs. 6 and 9.
For instance, in tumors treated with EGFR-TKI, cells start to overexpress αVβ3 integrin, leading to a resistant tumor; in fact, galectin-3 binds to the oligosaccharide moieties of β3 integrin and promotes integrin/KRAS interaction, independently of integrin-mediated adhesion to ECM proteins. KRAS activates the downstream RalB/NFkB pathway that leads to therapy resistance by promoting a stem cell-like phenotype,6 as shown in Figure 2b. Furthermore, a crosstalk between αVβ6 and EGFR, which regulates bidirectional force transmission and controls breast cancer invasion, have been recently described.10 Hence, therapeutic strategies targeting integrin/EGFR interaction to prevent the emergence of acquired resistance to EGFR-TKIs have been largely proposed.3
The integrin-GFRs crosstalk is important not only in the development of tumor resistance, but also in cancer progression, malignant transformation and metastasis spread. Increasing piece of evidence3 indicate that cells within the tumor environment upregulate the expression and signalling functions of integrins (mainly αVβ6 and αVβ3) inducing Epithelial-To-Mesenchymal Transition (EMT).
As described in Chapter 2 (Paragraph 2.1.2.), the cancer-related EMT process is characterized by transformation of cancer cells into invasive forms that migrate to other organs, leading to tumor progression and metastasis spread. For these reasons, targeting EMT-related biomolecules is emerging as a novel therapeutic approach for preventing migration and invasion in several cancer types.3,7 For instance, the combined administration of V integrin ligands and sunitinib has been shown to enhance the inhibitory effect of sunitinib on TGFβ1-induced EMT in human non-small cell lung cancer cells.7
To conclude, recent evidences show how the crosstalk of signaling pathways between integrins and growth factor receptors is extremely important in the development of tumor resistance and in
114 inducing EMT process, which aggravates cancer progression and leads to malignant transformation.
The development of dual agents aimed at inhibiting both integrins and GFRs simultaneously could be a promising therapeutic approach for tumor- and fibrosis-related diseases.
On this line, our research group has recently developed three novel dual covalent conjugates (3, 4 and 5, Figure 3) comprising the V3 integrin targeting cyclopeptidomimetic c(AmpRGD) (depicted in red), the TKI sunitinib moiety (depicted in blue), and three different types of linkers (depicted in black) with the aim to (i) exploit the c(AmpRGD) ligand ability to selectively target V3 integrin-overexpressing cells; (ii) enhance integrin-mediated cell internalization of the construct, (iii) possibly interfere with the crosstalk between V3 integrin and VEGFR, and (iv) address the sunitinib moiety at the respective intracellular TK targets.4,11,12
Figure 3. Structure of the three covalent conjugates synthesized in the last year by our research group.
The V3 targeting peptide moiety is represented in red, the linker spacer in black and the sunitinib unit in blue. The free V3 ligand c(AmpRGD)-NH2 (6) is displayed in the box.
In these compounds, conjugation of the integrin-recognizing peptide with the appended drug did not affect the ligand binding competence toward αVβ3; meanwhile, the kinase inhibitory activity of the constructs remained comparable to that of sunitinib alone.12 Conjugates 3–5 were studied in vitro (human melanoma cell lines M21 and A375, human ovarian cancer cell line IGROV-1) and in vivo (nude mice) as inhibitors of tumor angiogenesis and progression. It was proven that cell uptake was mediated by αVβ3 integrins, and dimeric compound 5 was better internalized as compared to
115 congeners 3 and 4, probably due to its enhanced affinity toward the integrin receptor and favourable physico-chemical properties (e.g. protonation state). It is worth noticing that dimeric compound 5 underwent only partial cell internalization and this behaviour was judged to be beneficial; the authors speculated that a possible synergy action could be operative involving both the extracellular RGD−integrin interaction (provided by the non-internalized fraction) and the sunitinib−VEGFR2 kinase interaction (provided by the amount of internalized compound).
Compounds 3 and 4, though structurally similar, showed a very different uptake profile and overall biological activity, highlighting how the structure of the linker could deeply influence the physical-chemical-biological properties of the resulting conjugates.
Interestingly, a decreased aggressiveness in tumor cell population was observed under chronic treatment with conjugates 3 and 5 as compared to sunitinib alone, opening the way for the use of these selective conjugates as drugs able to overcome the TKI-related tumor resistance. Finally, the in-vivo targeting ability together with tumor inhibition of compounds 3 and 5 was demonstrated by experiments on tumor implanted nude mice, indicating a striking antitumor activity of this conjugate versus sunitinib; taken together, these results support the interest of integrin-targeted sunitinib conjugates for the treatment of drug-resistant tumors.
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