Targeting tumour microenvironment by tyrosine kinase inhibitor

Protein phosphorylation, one of the most prevalent post-translational modification of protein, is tightly regulated by specific protein kinase that transfer phosphate group to the amino acid residue [1]. To our knowledge, there are in total 518 kinases in human genome [2], while 90 of them are classified in the category of tyrosine kinases (58 of them are receptor tyrosine kinase RTK; the remaining are non-receptor tyrosine kinase) [3]. Specifically, tyrosine kinase transfers phosphate group from ATP to tyrosine residue of the protein. In carcinogenesis, the aberrant activation of protein phosphorylation, particularly by RTKs has been frequently described [4]. Mutation or gene amplification of tyrosine kinase signalling further promote the carcinogenesis process, including survival, proliferation, motility, and metabolism as well as escape from immune surveillance [5]. For instances, overexpression of epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor α/β (PDGFR) have been well implicated in supporting various malignancy growth and progression [6, 7].

The binding of ligand such as growth factors or cytokines to the extracellular domains of RTKs initiate the signalling cascade by changing its structure and kinase activation. Whereas the non-RTKs which lack of extracellular domains mainly serves as the downstream effector of RTKs [8]. Understanding on the mode of action of tyrosine kinases had led to discovery of many small molecule inhibitors proved to be effective for the cancer treatment. To date, more than 30 RTK inhibitors have been approved by US FDA. They can inhibit single target or multiple targets. For instances, gefitinib and erlotinib which primarily inhibit EGFR, is used for EGFR-mutated lung cancer patients [9]. Imatinib mesylate has multiple targets, c-KIT, PDGFR and c-ABL, is indicated for acute and chronic myeloid leukemic as well as gastrointestinal stromal patients [10, 11]; sorafenib which multi-targeted VEGFR, PDGFR and Raf, is utilized for advanced renal cell carcinoma and hepatocellular carcinoma patients [12, 13]. Much previous efforts on tyrosine kinase inhibitors (TKIs) have focused on their direct actions in regulating tumour growth and angiogenesis, while recent emerging studies have re-focused on how TKIs modulate the tumour microenvironment (TME). An appropriate understanding on how TKIs alter the TME may help pave another mode of action of TKIs in cancer therapy. To maximally exploit the therapeutic benefit of current tyrosine kinase inhibitors, we systematically searched through the PubMed database with the MeSH terms of “tumour microenvironment” and “Protein-Tyrosine Kinases/antagonists and inhibitors”. In this review, we highlight the impact of kinase activation and paracrine production of ligand from tumour stroma, and summarise the findings on the potential effect of various type of tyrosine kinase inhibitors on tumour microenvironment components which including mesenchymal cells, hematopoietic cells and non-cellular components. The perspective of combination use of current kinase inhibitor with immunotherapy and modulation of TME in overcoming TKIs resistance for cancer treatment are also discussed.

Kinase inhibition in tumour cells by tyrosine kinase inhibitors offers promising clinical benefit to cancer patients, especially, who have tumour with mutated kinases [14]. Nonetheless, aberrant activation of tumour microenvironment may fail therapeutics that are merely targeting on cancer cells. The fact that tumour cells can promote their growth by recruiting and communicating with other type of cells, such as mesenchymal- and hematopoietic-originated cells, in the tumour microenvironment (TME) [15]. Given the accumulating evidences of tyrosine kinase paracrine receptor activation or ligand production by TME components, TKIs may be a promising TME modulator apart from regulating the intrinsic functions of tumour cell [16‐18]. Taken the example of tumour immunology, TKIs generally act on the two mechanisms: immunogenic control and immune conditioning [19]. Immunogenic control is the modulation of tumour cells sensitivity in response to alteration of immunosuppressive phenotype, whereas immune conditioning is the direct changes of TKIs incurred on the function and number of immune cells populations. As detailed below, TKIs have been implicated in essentially all components of TME and thus increasingly recognised as a promising candidate for TME modulation.

Extracellular matrix ECM components including fibronectin, laminin and collagen have been shown to determine the fate of cancer cells whether to remain dormant or proliferative [139]. It was demonstrated that collagen or fibronectin accumulation promoted progression of dormant breast cancer cells via β1-integrin mediated SRC and FAK activation. SFK inhibition with AZD0530 offered transient blockade on proliferation of dormant cancer cells, and only combination of AZD0530 with selumetinib, MEK inhibitor prevented the dormant-to-proliferative shift of cancer cells [140]. Furthermore, the composition of ECM also governs the therapeutic outcome of cancer treatment. Earlier study postulated that small cell lung cancer cells tend to accumulate at ECM rich region, whereby the ECM proteins further protected the cancer cells from chemotherapy. The adhesion of cancer cells to fibronectin triggered activation of protein tyrosine kinase and intervention with tyrphostin-25, a competitive EGFR tyrosine kinase inhibitor abolished the effect [141]. The similar phenomenon has also been documented by another two potent TKIs genistein and tyrophostin AG-1478 [142].

It has been proposed in various types of human cancers that immunotherapeutic regimen could offer favourable outcome, though large-scale randomized, placebo-controlled trials shall be carried out prior to its application. Given the accumulating reports on acquired resistance of TKIs, the use of TKIs alone as first-line treatment seems challenging in spite of its better prognosis. TKIs treatment often gain rapid but not durable tumour response, which result in early improvement of survival curve of cancer patients without clear beneficial effect on the overall survival. The effect of TKIs could be strategically complemented by immunotherapy, which induces a low percentage but highly durable tumour response [143]. This notion has been partially supported by pre-clinical observation in mouse model, which showed that an ALK vaccine improved tumour relapses after TKI suspension [144]. The idea combining TKIs with immunotherapy raised in early 2010s and some efforts, in both pre-clinical and clinical extent, have been made to investigate its beneficial effect in cancer treatment [145]. Although most of the studies are still on-going, progress report in recent conference proceedings have suggested the prominent potential of this brilliant combination. For instance, McDermott et al. reported that combination of Atezolizumab (anti-PD-L1) with sunitinib (VEGFR inhibitor) resulted in encouraging anti-tumour effect than TKIs alone in metastatic renal cell carcinoma (mRCC), and a large scale phase III trial is highly suggested [146]. On-going studies from Ribas group suggested combination of pembrolizumab (anti-PD1) with dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) for phase II study in BRAF-mutant melanoma patients after manageable toxic profile of this combination has been observed [147]. Mechanistically, the action of combination treatment may be in sequential or parallel. For instances, sequential use of sorafenib followed by sunitinb improved overall survival of the metastatic renal cell carcinoma patients, suggesting the sequential action of this combination [148]. However, given the regulation of TKIs on tumour microenvironment as we recognized in this review, it has been expected that the combination treatment would deliver a parallel action, that is, TKIs and immunotherapy synergize each other. It was observed that combination of anti-angiogenic TKIs sorafenib/sunitinib with anti-tumour rMVA–CEA–TRICOM vaccine, could synergistically increase the CD8+ tumour infiltrating lymphocytes and intratumoral CD11b + cells, which is hardly observed in TKIs or vaccine treatment alone. This is partially related to the regulation of TKIs alone on the phenotypes of immunosuppressive MDSCs and TAMs [149]. Combination of TKIs with FLT3-directed immunotherapy enhanced its therapeutic outcome on acute myeloid leukaemia, which is probably due to the induced expression and localization of FLT3 on the cell surface [150]. Efficacy and mechanism of action of combination treatment using TKIs and immunotherapy are expected to be under more extensive inspection.

Furthermore, safety of the combination use is still contradicting and unclear. Some studies have reported the increased risk of adverse reaction after combination therapy, including higher incidence of grade 3/4 liver enzyme elevation (40–70%) and high incidence of interstitial lung disease (38%) [151]. A lot of recent efforts were thus made to justify the safety use of combination treatment. A phase I study on the safety of combination treatment of cobimetinib (MEK inhibitor) and atezolizumab (anti-PD1) showed that the well tolerance of colorectal cancer patients at the maximum administered doses [152]. Combination of atezolizumab (anti-PD1) with vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor) induced rash and elevated liver enzymes in patents with BRAF-mutant melanoma, however, the adverse reaction was manageable by a run-in period of vemurafenib and cobimetinib [153]. However, unsuitability of combination treatment between immunotherapy and TKIs was also reported. For instances, combination of pazopanib (TKIs) and pembrolizumab (anti-PD1) resulted in significant hepatotoxicity in advanced RCC patients. While sequential regimen showed improved efficacy yet limited tolerability [154]. Interestingly, it was somewhat suggested that combination treatment may improve the adverse reaction, which may be probably due to the dose-reduction effect in TKIs. Bhatia et al. reported a favoured outcome in clinical trials using combination of IL21 with sorafenib to treat mRCC. The combination use of IL21 resulted in appropriate dose-reduction in sorafenib associated with improved adverse reaction [155]. These pilot findings suggested the safety of combinatory treatment of immunotherapy and TKIs on cancer patients warrant continued exploration for clinical benefit.

The development of acquired resistance remain as the major clinical challenge to TKIs treatment, despite TKIs are still the first line therapeutic regimen for many malignancies. For instances, non-small cell lung cancer patients who initially well responded to EGFR inhibitors, erlotinib or gefitinib had host adaptive response and tumor growth within 6–12 months [156, 157]. Majority of the first line TKIs aiming at the intrinsic function of tumor cells, while neglecting that the susceptibility of tumor cells may shift in response to the alteration of microenvironment. In fact, upon TKIs perturbation, the tumor microenvironment compartment tends to secrete the extrinsic factors such as cytokines, hormones, or growth factors in sensitizing or de-sensitizing the response of cancer cells. This notion was further supported by pre-clinical observations that the paracrine secretions of various growth factors by neighboring fibroblast such as hepatocyte growth factors (HGF) and neuregulin 1 (NRG1) further promoted cancer cell resistance to TKIs [60, 158]. While re-sensitization of cancer cells to TKIs is often achieved by either suppressing the secretive factors by TME or antagonizing the receptors on cancer cells. It was observed that co-administration of EGFR inhibitors with apatinib, a highly selective VEGFR2 inhibitor reduced the expression of VEGF in TME, which further delayed tumor growth in mice and prolonged disease-free survival in cancer patients [159]. The fibroblast and endothelial cells produced HGF and EGFR ligands reduced the cancer cell response to crizotinib, a dual inhibitor of ALK and Met. While co-administration of EGFR inhibitors enhanced the susceptibility of cancer cells to crizotinib [61]. These attempts suggest that manipulating TME by repressing pro-tumoral secretive factors or blocking receptors on cancer cells may be beneficial in overcoming TKIs resistance.

Also, it is believed that cancer cells develop an immunosuppressive microenvironment for evasion from immune surveillance upon TKIs intervention. Although the detailed mechanisms of acquired immune evasion is not yet understood, previous studies suggested that the inducible expression of immune checkpoints PD-1/PD-L1 may be involved. Notably, the elevated expression of PD-L1 was observed in EGFR-resistant tumor biopsy compared to pre-treated tumor samples. This suggests the activation of immune checkpoint may hinder the treatment efficacy of TKIs to cancer patients [160]. Furthermore, co-treatment of immune checkpoint inhibitor, nivolumab in lung cancer patients after disease progression with TKIs showed improved progression-free survival and the enhanced progression-free survival rate was in proportion to PD-L1 expression [161]. Similar observation was obtained from patients receiving ALK inhibitors, whereby anti-PD-1 immunotherapy restored the efficacy of ALK-targeted therapy [144]. Therefore, the treatment strategies aim at normalizing or reprogramming the immunosuppressive cancer environment may be promising in reversing TKIs resistance and achieve a favorable outcome of TKIs-associated cancer therapy.

With recognition of the actions of TKIs on TME, our previous studies have also suggested the beneficial role of complementing TKIs with immune-modulatory therapy. Complementary treatment using PHY906, a composite herbal formula that had potential to increase the therapeutic index of cancer treatments in multiple clinical trials, was recently identified to modulate TME in sorafenib-treated hepatocellular carcinoma (HCC). This immune-modulatory effect of PHY906 improved the anti-tumour TME signature and thereby enhanced the efficacy of sorafenib [162]. An active component isolated from PHY906, named baicalin, has evidenced to contribute to this immuno-modulatory action by specifically targeting on autophagic pathway in TAMs [163]. These observations combined with our study on other natural molecules suggest the potential of using immuno-modulatory complementary therapy [164] in combination with TKIs as emerging cancer therapy.

In past decade, tyrosine kinase inhibitors targeting at tumour cells represent a promising therapeutic agent for cancer patients; yet, the use of TKIs in modulating tumour microenvironment is still in its infancy. The recent advances we summarized here emphasize the potential use of TKIs for re-educating or normalizing the dysregulated tumour microenvironment for cancer treatment. An understanding of the modulatory effect of TKIs on tumour microenvironment could expedite the maximal use of TKIs on cancer therapy as well as re-positions the existing TKIs as combination cancer treatment. Furthermore, phosphorylated status of target protein could also be determined by protein phosphatase, which belongs to another group of protein involved in microenvironment status of tumour cells, is worthy for further exploration. Much more efforts, either pre-clinically or clinically, is therefore anticipated in order to maximize the efficacy and safety for patients.