It is well known that breast cancer is the most common cause of death in women[13]. There are various treatment therapeutic regimen,including targeted therapy and immunotherapy. Apatinib has a good antitumor effect in breast cancer[14]. Correspondingly, cancer cell will death when researcher block the PD-1/PD-L1 by activating tumor cell-intrinsic signaling[15]. However, The effective rate of single drug is only about 20%[16]. There have clinical studies shown that the combination of immunotherapy and targeted therapy has a good effect on tumor treatment. Zhang yu[17] suggested that apatinib combined with PD-L1 blocking therapy could synergistically enhance the anti-tumor immune response in GC. And in liver cancer,the control rate of advanced liver cancer is 93.3% through the combination of the two drugs[18]. This combination can extend the benefit of PD-L1 inhibitor treatment to a larger population of breast cancer.
In this study, we demonstrate a critical role of PD-L1 inhibitor and apatinib in conferring breast cancer with a variety level of studies. We find PD-L1 expressed in both MCF-7 and MDA-MB-231, while PD-L1 expressed stronger in MDA-MB-231 than in MCF-7 (Supplementary 1). Then, we showed that the targeted drug apatinib synergized with PD-L1 inhibitor against breast cancer in vitro and in vivo. MTT assay showed that PD-L1 inhibitor significantly inhibited the cell viality of the breast cancer MCF-7 and MDA-MB-231 cell line in a concentration-dependent manner. The same conclusion is true for apatinib. At the same time, a large number of studies have proved that apatinib[19]and PD-L1 inhibitor[20]can inhibit the occurrence and development of MCF-7 and MDA-MB-231 cells.we observed increased antitumor efficacy when apatinib was combined with anti–PD-L1 in the in vitro. Samely,Sha Zhao[21]think that apatinib can potentiates antitumor effect of PD-L1 blockade in cancer. In the synergistic experiment, the combination of the two drugs plays a synergistic effect in the treatment of cancer. Moreover, We find that apatinib sequential PD-L1 inhibitor have better effect than the other two combination mode. There are studies that apatinib combined with PD-L1 blockade synergistically enhances antitumor immune responses[22, 23]. In the invasion and metastasis experiment, Our study showed that apatinib and PD-L1 inhibitor significantly inhibited cell migration and invasion in the breast cancer cells. We observed different therapeutic effects with different concentrations of the drug. When the drug concentration increases, the invasion and metastasis ability of cells decreases. Compared with the single drug group, the number of cells invading the lower chamber in the combined group decreased by about twice. In Haige Zhang ‘s study,apatinib can suppress proliferation and invasion of the cancer cells via angiomotin inhibition[24]. Samely, a Phase 1B JAVELIN solid tumor study achieve expectations with acceptable side effects in metastatic breast cancer[25]. The effects of apatinib and PD-L1 inhibitor mainly occurred in the early stage of apoptosis on MCF-7 cells. While the result of drugs on MDA-MB-231 cells mainly focused on the late stage of apoptosis. We suggest that apatinib may induce apoptosis in cells by PI3K/AKT or MAPK/ERK signaling pathways. Then, we find that apatinib can also triggers apoptotic cell death via influencing PD-L1 signaling in cancer[26]. In a certain dose range, with the increase of concentration, the depressor effect of the drug is stronger. And again, we found that apatinib Combined with PD-L1 inhibitor synergistically enhances antitumor immune responses in other malignant tumor[23, 24].
To gain insight into the potential molecular mechanisms and signaling pathways involved in the synergistic anticancer effects in breast cancer cell line, we performed the western blotting to evaluate molecular changes upon mono or combination therapy. Apatinib and PD-L1 inhibitor through downregulated p-Erk and reduced the protein level of NF-κB in the breast cancer cells, and then downregulated its regulatory target Slug, therepy inhibit cell invasion and metastasis. The inhibition of p-Erk, NF-κB and Slug at lower concentrations was not obvious. In parallel, the inhibition of signaling pathways was most significant when combined drug therapy was used. Consistent with the result of Zhou[27] et al. Our results suggest that PD-L1 inhibitors and apatinib can inhibit Erk expression in breast cancer in a concentration-dependent manner. Recent evidenced have shown that the mechanisms of PD-L1 inhibitor were related with a variety of signaling pathways and specific molecular mechanism[28, 29]. The antitumor effects of apatinib reportedly have been reported at downstream of the VEGFR2 signaling pathway via PI3K/AKT/mTOR or MAPK/ERK[30]. Mechanistically, PD-L1 prominently activated epithelial mesenchymal transition (EMT) process through the MEK/Erk signaling pathway[31]. In this study, PD-L1 inherent in cancer cells can also promote the transduction of intracellular signaling pathway Ras/p-Erk/slug[31]. More deeply, apatinib can weaken the expression of PD-L1 by inhibiting the ERK signaling pathway. However, Inhibition of activation of ERK was a key point for suppression of NF-κB/p65 modulated metastatic mechanism in Jin's study[32]. The inhibition of NF-κB/p65 can take the edge off the slug and reverts the EMT expression pattern[33]. The Slug may be involved in the resistance of epithelial cells to apoptosis and promote tumor invasion through some mechanisms[34],and is regulating the invasion and growth of breast cancer[35]. Consistent with prior study, PD-L1 overexpression or knockout affects the proliferation and migration of cancer cells through Erk signaling pathway[36]. Meanwhile, MAPK/ERK signaling pathway can affect the expression of PD-L1[37, 38]. Therefore, these findings indicate that combination of apatinib and PD-L1 inhibitor can inhibit cell proliferation, reduce migration and invasion ,increase apoptisis possibly by attenuating the expression of p-Erk/NF-κB/Slug in breast cancer cells.
To confirm this synergy on tumor cells, we also test the effects of apatinib and PD-L1 inhibitor in vivo. The results indicated that the anticancer effect of the combinatorial treatment was higher than any other single drug, which was consistent with in vitro. In addition, nude mices were treated with the combinatorial treatment did not show worse body weights than the patients in the groups treated with apatinib or PD-L1 inhibitor alone. It indicates that the drug toxicity is within the acceptable range. Hence, our data show that a combination of apatinib and PD-L1 inhibitor led to enhanced antitumor activity. The studies from Schmittnaegel[39] evidenced that antiangiogenic agents could improve anti–PD-L1 therapy in various tumor models. The Combination of antiangiogenic and anti–PD-L1 therapy can stimulate Body tumor immunity in a previous study[40]. There were studies that demonstrated the effect of apatinib in combination with PD-L1 mAb from another perspective[41]. There are a closing correlation between targeted therapy and immunotherapy in cancer[42]. Similarly, apatinib enhances anti-tumor efficacy when combined with immunotherapy in preclinical studies[40, 43]. Apatinib not only inhibits angiogenesis, but also affects PD-L1, thereby mediating antitumor immunity. Nevertheless, We need to conduct more experiments to confirm the clinical efficacy of apatinib combined with PD-L1 inhibitor on breast cancer.
In summary, this work reveal the antitumor activity of apatinib combined with PD-L1 inhibitor on breast cancer in vitro and in vivo. The study supposed that the interaction of apatinib and PD-L1 inhibitor induced the activation of p-Erk/NF-κB/Slug cascade, thus promoting Breast cancer cell migration/invasion (Fig. 6). It provides partial theoretical basis for the combination of targeted therapy and immunotherapy for breast cancer. However, there are many challenges remain to be overcome before the potential of The combination therapy can be realized.