Treatment Strategies of Gastric Cancer—Molecular Targets for Anti-angiogenic Therapy: a State-of-the-art Review

VEGF is one of the most significant factors involved in the stimulation of angiogenesis during gastric carcinogenesis [37]. The VEGF family consists of 7 major subtypes including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PIGF [38, 39]. VEGF-A released by the gastric cancer cells is considered to be the major pro-angiogenic factor involved in gastric tumorigenesis [40]. Moreover, the expression of VEGF-A increases the number of vessels in both - the intestinal and the diffuse type of GC [41]. Studies demonstrated a worse overall prognosis in patients with VEGF-positive tumours compared to the VEGF-negative ones [42].

The binding of VEGF-A to its receptor—VEGFR-2—is considered to be the most crucial activator of the angiogenic pathways [43]. The binding promotes a cascade of signals that result in the proliferation and migration of the ECs, enhanced vascular permeability, modification of gene expression, and activation of the Ras pathway [44, 45]. Contrarily, the role of VEGFR-1 is more intricate and not fully deciphered yet. A soluble form of VEGFR-1 can act as a bait receptor and prevent VEGF-A/VEGFR-2 binding, which inhibits the activation of the signalling pathway. However, there is evidence that VEGFR-1 plays an essential role in the progression of angiogenesis [46]. VEGFR-3 stimulates lymphangiogenesis and does not bind to the VEGF-A [47].

The neuropilins 1 and 2 (NRP1 and NRP2) are involved in the cell guidance and the enhanced binding of VEGF to its signalling receptors [48]. A vast number of other factors present similarity to VEGF-A in terms of the stimulation of the angiogenesis and those include PIGF, FGF, VEGF-C, VEGF-D, angiopoietin, HIF-1α and HIF-2α, integrins, and PDGF [49, 50].

Overexpression of another member of the VEGF family—PIGF—leads to pathological angiogenesis [51]. Furthermore, it was shown that PIGF overexpression correlates with metastases to lymph nodes, tumour growth, and poor overall survival in GC [52].

Resistin-like-molecule-α (RELM-α) is a protein that plays a significant role in GC progression despite being a marker for anti-inflammatory macrophages [53]. It was reported that RELM-α expression is associated with tumour size and staging [54]. Besides, RELM-α activates the members of the VEGF family by activating the NF-κB-MMP-9/VEGF pathway, enhancing the process of angiogenesis [55]. RELM-α is now considered a new and promising biomarker in GC [5].

During physiological conditions, angiopoietins (Angs) are involved in the embryonic and postnatal angiogenic processes. So far, the most examined members of this family include angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), which are highly overexpressed in gastric cancerous tissues [56]. Ang-1 and Ang-2 are the ligands for the tyrosine-protein kinase receptor—Tie-2. Ang-1/Tie-2 binding stabilizes the vessels due to the recruitment of the pericytes [57]. On the contrary, Ang-2 antagonizes Tie-2 preventing the maturation of Ang-1, which leads to the suppression of vasculature growth [58]. Furthermore, the disparities between Ang-1 and Ang-2 levels might be associated with the severity of the pro-angiogenic process.

PDGF-β is a crucial component of the tumour progression, as it induces both angiogenesis and EMT, which are highly relevant regarding the alterations within the GC microenvironment [59]. It was shown that PDGF-β and VEGF are secreted simultaneously in gastric cancerous tissues, though PDGF-β is believed to be more crucial in the maintenance of the blood vessels in the intestinal type of GC [60]. In addition, PDGF-β is essential in the migration, proliferation, and adhesion of the endothelial progenitor cells, which are required for both neovascularization and re-endothelialization [61, 62].

IL-8 was originally described as a chemokine whose main role was to attract the polymorphonuclear inflammatory leukocyte infiltrate acting on CXCR1/2 [63]. Recently, it was found that various tumours often overproduce this chemokine, which presents the pro-tumoural characteristics in various malignancies [64]. IL-8 is involved in the angiogenesis, survival signalling for cancer stem cells, and attraction of the myeloid cells, as well as delivery of the local growth factors [65]. Moreover, IL-8 is highly produced by the tumour cells; thus, its serum concentration is significantly associated with tumour growth [66]. Therefore, IL-8 serum concentration is considered to be a useful pharmacodynamic biomarker in the early detection of the immunotherapy response in GC patients [67].

MicroRNAs (miRNAs) constitute a group of small, single-stranded, non-coding RNAs that regulate the expression of various genes at the point of post-transcription [68]. Hence, miRNAs are involved in various cellular functions such as proliferation, apoptosis, regulation of embryonic stem cell advancement, and cancer cell invasion [69]. The latest studies have shown that miRNAs are remarkably stable in the blood flow, besides their concentration might present the efficacy of the therapy; this discovery shows that they might be reliable biomarkers of GC therapy [70]. Several studies showed that miRNAs can regulate tumour angiogenesis through targeting both the pro- and antiangiogenic factors, such as RTK signalling protein, HIF, VEGF, and epidermal growth factor (EGF) [71]. miRNA-126 has an essential role in regulating the angiogenesis processes; elevated expression of miRNA-126 is associated with the increased VEGF-A signalling in ECs. Therefore, miRNA is believed to be a promising biomarker of the anti-angiogenic therapy of GC [72, 73].

Circulating tumour cells (CTCs) and free nucleic acid (CTNA) are metastatic cells that are released into the bloodstream by the primary tumour cells; thus, they are involved in the recognition of the hematogenous metastasis [74]. CTCs and CTNA detection in the peripheral blood is a potential strategy for the non-invasive diagnosis and estimation of GC patients’ response to treatment therapies [75, 76]. The conducted studies suggest that patients with a low baseline CTC count or drop of the CTC amounts after the first cycle of chemotherapy might substantially benefit from the palliative chemotherapy [77]. Therefore, CTC count might be a good chemotherapy-supervising marker and a prognostic marker for patients receiving palliative chemotherapy [49].

The surgical treatment of GC constitutes the major and most frequently used treatment strategy in GC patients; two major types—open and minimally invasive laparoscopic gastrectomies—are distinguished [81, 82]. Laparoscopic gastrectomy can be performed by either the operator or a robot—so-called robotic laparoscopy [81]. The Japanese Gastric Cancer Association distinguishes several types of gastrectomies including total gastrectomy, distal gastrectomy, pylorus-preserving gastrectomy, proximal gastrectomy, segmental gastrectomy, local resection, and non-resectional surgery such as bypass surgery or gastrostomy [82]. Best et al. [83] showed no difference in the short-term mortality rates between laparoscopic and open gastrectomies. The laparoscopic treatment includes the totally laparoscopic distal gastrectomy (TLDG) and the laparoscopic-assisted distal gastrectomy (LADG). Hyui et al. [84] showed that there is no remarkable difference in surgical outcomes and postoperative complications between TLDG and LADG. It was reported that TLDG was preferably chosen due to the lower percentage of postoperative pain and quicker recovery. Surgery treatment also includes the dissection of the lymph nodes [85]. Regional lymph nodes of the stomach are classified into the stations from 1 to 20 plus stations 110, 111, and 112 [86]. The nodal resection as a part of GC treatment can be divided by the D-level criteria depending on the type of gastrectomy [82, 86]. The extent of lymphadenectomies performed worldwide differs significantly between the countries [87].

Chemotherapy, as a perioperative treatment, has been improved significantly over the recent years [88]. The survival rate of patients treated with chemotherapy increased compared to patients treated applying the surgical resection alone [89, 90]. In the case of GC, chemotherapy is preferred in patients with AGC unlike in the case of patients with EGC patients, who are preferred to be treated applying surgical methods [91]. Usually, chemotherapy does not provide a complete cure, however, in the case of patients with unresectable GC, the obtained median survival time was estimated at 6–13 months; adjuvant chemotherapy also seems to be reasonable [82]. Macdonald et al. [92] compared surgical treatment alone versus surgical treatment with fluorouracil and leucovorin treatment combined with radiotherapy. Eventually, the overall survival rate while applying only surgery alone was 26 months, and in investigated adjuvant chemoradiotherapy, it was prolonged up to 36 months [92]. Also, Guimbaud et al. [93] in phase III of their prospective, multicenter, randomized trial compared epirubicin, cisplatin, and capecitabine (ECX) to fluorouracil, leucovorin, and irinotecan (FOLFIRI) as the first-line treatment of AGC and gastroesophageal junction (GEJ). After a median of 31 months, follow-up time-to-treatment failure (TTF) was significantly longer for FOLFIRI in comparison to TTF of ECX (5.1 versus 4.2 months) [93]. The authors announced that FOLFIRI as first-line treatment should be considered as a backbone regiment for targeted treatment agents and, in this case, should be explored [93]. Surgical treatment and dissection of the tumour can cause the activation of the tumour cell growth–stimulating factor and lead to immediate growth of GC tumour. Moreover, it can entail the production of anti-chemotherapy agents [94]. Thus, the target of neoadjuvant chemotherapy (NAC) is to down-stage tumour and to eliminate potential metastases which can let R0 resection [79]. NAC as a worldwide-accepted treatment was approved by MAGIC randomized trial 903. Terashima et al. [95] in their phase III randomized trial take on targeting the efficiency of NAC considering morbidity, morality, and surgical aspect in stage 3 and stage 4 GC. In their results, there was no major growth of morbidity and mortality registered; however, the time of surgical procedure was significantly shorter (median time equal to 240 and 255 min for those with and without NAC, respectively) [95].

Radiotherapy is a treatment modality that is frequently combined with chemotherapy in the so-called chemoradiotherapy. After the American SWOG/INT0116 clinical trial, radiotherapy currently constitutes a standard treatment strategy just after the radical gastrectomy in many Oncological Units [96, 97]. However, radiotherapy is also developed as a palliative, first in the 1960s, and adjuvant to neoadjuvant treatment of GC [78, 98]. Patients with not fully resected tumours are treated palliatively. The recurrent or locally advanced tumour treated by radiotherapy does not give a well-tolerated modality to palliate bleeding, obstruction, or pain, but it is also effective [98]. There are few methods of radiotherapy such as proton beam radiotherapy (PBT) or photon radiotherapy (RT) [98]. There are also some techniques of treatment simulation such as 2-dimensional simulation or 3-dimensional simulation [99, 100]. During the radiation therapy treatment, patients are exposed to 45 to 50.4 Grey (Gy) in total, which can eventually result in serious adverse reactions [78, 101]. Gao et al. [102] showed that intraoperative radiotherapy (OIRT) did not prolong the overall survival of GC patients but instead, and it had favourable effects in stage II and stage III tumours.

Immunotherapy with checkpoint inhibitors is a novel method that has entered the clinical practice as the human immune system can be stimulated to identify the malignant tumours by the immune surveillance and ultimately inhibit the tumour growth [103, 104]. Nowadays, immunotherapy is targeted at the expression of the PD-1 checkpoint receptor ligand, PD-L1, and the tumours with microsatellite instability (MSI-H) [103, 105]. PD-1 is expressed on the activated T-lymphocytes (primarily to the intra-tumoural antigen–specific CD8+ T lymphocytes), and binds its ligands—PD-L1 and PD-L2—on the tumour cells which leads to exhaustion, anergy, or apoptosis of that lymphocyte and let the growth and development of the tumour [106‐110]. Thus, blocking those immune checkpoints stimulates the T lymphocytes against GC cancer cells by avoiding their binding to the ligands [106]. In the USA, the first and only inhibitors approved by the Food and Drug Administration (FDA) in MSI-H tumours are pembrolizumab and nivolumab (primarily used in Japan). Several others are currently investigated in the clinical trials—ramucirumab (phase I), leucovorin (phase II), avelumab (phase III); the last one was accepted by the European Medicines Agency (EMA) [111]. According to Kamath et al. [106], pembrolizumab provided a promising response at about 12–22% in third-line treatment of locally advanced and metastatic GC.

Molecularly targeted therapy is a treatment modality that is based on the growth, cell cycle, apoptosis, angiogenesis, and invasion [78, 112, 113]. Main treatment strategies include the epidermal growth factor receptor (EGFR), VEGF, matrix metalloproteinase (MMP), erythroblastic leukemia viral oncogene homolog 2 (HER2—a member of the EGFR family), and RTK/RAS pathway [78, 80, 114].

EGFR is the receptor whose activation stimulates the intracellular pathways that lead to enhanced proliferation, migration, and angiogenesis. Thus, blocking this receptor inhibits tumour proliferation [78]. There are several agents that block EGFR such as anti-EGFR monoclonal antibodies or EGFR tyrosine kinase inhibitors (EGFR-TKI) [78]. Cetuximab is considered to be a good medicament in the untreated GC [115]. Pinto et al. [115] in their phase II trial compared cetuximab plus docetaxel and cisplatin to docetaxel and cisplatin alone. The rate of response was 41%, and it was higher than in docetaxel and cisplatin alone treatment [115, 116].

VEGF belongs to the family of cytokines that regulate angiogenesis [117]. This family consists of 7 main subtypes including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF [118]. There were two clinical phase III trials—REGARD and RAINBOW—both focused on ramucirumab (VEGFR-2 antibody) on previously treated AGC [119, 120]. However, despite the promising results, those agents do not inhibit the progression of GC; however, they can prolong survival time, unfortunately, only in months [117].

The MMP family consists of few members, inter alia MMP-1, -2, -7, -9, and -12 [78]. MMPs degrade cellular compounds, which in consequence leads to growth, progression, and metastases of the tumour [121]. Marimastat is the MMP inhibitor of the abovementioned MMPs; combined with 5-fluorouracil (5-FU), this drug is considered to be a valuable anticancer treatment strategy [122].

An HER-2 signalling pathway is another way of treatment that is continually investigated and improved [114]. There are few ongoing trials (phase II and III) of trastuzumab: NCT02205047 (INNOVATION)—phase II; NCT01774786 (JACOB)—phase II; and RTOG 1010—phase III. Trastuzumab-based therapies are continually improved especially in the case of HER2-positive GCs [114, 123].