Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, São Paulo, Brazil.
Pharmacol Res. 2016 Sep;111:523-533. doi: 10.1016/j.phrs.2016.07.017. Epub 2016 Jul 18.
The BRAF(V600E) mutation confers constitutive kinase activity and accounts for >90% of BRAF mutations in melanoma. This genetic alteration is a current therapeutic target; however, the antitumorigenic effects of the BRAF(V600E) inhibitor vemurafenib are short-lived and the majority of patients present tumor relapse in a short period after treatment. Characterization of vemurafenib resistance has been essential to the efficacy of next generation therapeutic strategies. Herein, we found that acute BRAF inhibition induced a decrease in active MMP-2, MT1-MMP and MMP-9, but did not modulate the metalloproteinase inhibitors TIMP-2 or RECK in naïve melanoma cells. In vemurafenib-resistant melanoma cells, we observed a lower growth rate and an increase in EGFR phosphorylation followed by the recovery of active MMP-2 expression, a mediator of cancer metastasis. Furthermore, we found a different profile of MMP inhibitor expression, characterized by TIMP-2 downregulation and RECK upregulation. In a 3D spheroid model, the invasion index of vemurafenib-resistant melanoma cells was more evident than in its non-resistant counterpart. We confirmed this pattern in a matrigel invasion assay and demonstrated that use of a matrix metalloproteinase inhibitor reduced the invasion of vemurafenib resistant melanoma cells but not drug naïve cells. Moreover, we did not observe a delimited group of cells invading the dermis in vemurafenib-resistant melanoma cells present in a reconstructed skin model. The same MMP-2 and RECK upregulation profile was found in this 3D skin model containing vemurafenib-resistant melanoma cells. Acute vemurafenib treatment induces the disorganization of collagen fibers and consequently, extracellular matrix remodeling, with this pattern observed even after the acquisition of resistance. Altogether, our data suggest that resistance to vemurafenib induces significant changes in the tumor microenvironment mainly by MMP-2 upregulation, with a corresponding increase in cell invasiveness.
BRAF(V600E) 突变赋予了组成性激酶活性，占黑色素瘤中 BRAF 突变的 >90%。这种遗传改变是目前的治疗靶点；然而，BRAF(V600E)抑制剂 vemurafenib 的抗肿瘤作用是短暂的，大多数患者在治疗后短时间内出现肿瘤复发。vemurafenib 耐药性的特征对于下一代治疗策略的疗效至关重要。在此，我们发现急性 BRAF 抑制诱导活性 MMP-2、MT1-MMP 和 MMP-9 的减少，但不会调节 naïve 黑色素瘤细胞中的金属蛋白酶抑制剂 TIMP-2 或 RECK。在 vemurafenib 耐药的黑色素瘤细胞中，我们观察到生长速度较慢，EGFR 磷酸化增加，随后活性 MMP-2 表达恢复，这是癌症转移的介质。此外，我们发现 MMP 抑制剂表达的不同谱，其特征是 TIMP-2 下调和 RECK 上调。在 3D 球体模型中，vemurafenib 耐药黑色素瘤细胞的侵袭指数比其非耐药对应物更为明显。我们在 Matrigel 侵袭实验中证实了这种模式，并表明使用基质金属蛋白酶抑制剂可降低 vemurafenib 耐药黑色素瘤细胞的侵袭，但不能降低药物-naive 细胞的侵袭。此外，我们在重建皮肤模型中没有观察到vemurafenib 耐药黑色素瘤细胞中有一组限定的细胞浸润真皮。在含有 vemurafenib 耐药黑色素瘤细胞的 3D 皮肤模型中发现了相同的 MMP-2 和 RECK 上调谱。急性 vemurafenib 治疗诱导胶原纤维的紊乱，从而导致细胞外基质重塑，即使在获得耐药性后也观察到这种模式。总之，我们的数据表明，vemurafenib 耐药诱导肿瘤微环境发生重大变化，主要是通过 MMP-2 上调，相应地增加细胞侵袭性。
