Mek inhibition results in marked antitumor activity against metastatic melanoma patient-derived melanospheres and in melanosphere-generated xenografts

Abstract

One of the key oncogenic pathways involved in melanoma aggressiveness, development and progression is the RAS/BRAF/MEK pathway, whose alterations are found in most patients. These molecular anomalies are promising targets for more effective anti-cancer therapies. Some Mek inhibitors showed promising antitumor activity, although schedules and doses associated with low systemic toxicity need to be defined. In addition, it is now accepted that cancers can arise from and be maintained by the cancer stem cells (CSC) or tumor-initiating cells (TIC), commonly expanded in vitro as tumorspheres from several solid tumors, including melanoma (melanospheres). Here, we investigated the potential targeting of MEK pathway by exploiting highly reliable in vitro and in vivo pre-clinical models of melanomas based on melanospheres, as melanoma initiating cells (MIC) surrogates. MEK inhibition, through PD0325901, provided a successful strategy to affect survival of mutated-BRAF melanospheres and growth of wild type-BRAF melanospheres. A marked citotoxicity was observed in differentated melanoma cells regardless BRAF mutational status. PD0325901 treatment, dramatically inhibited growth of melanosphere-generated xenografts and determined impaired tumor vascularization of both mutated- and wild type-BRAF tumors, in the absence of mice toxicity. These results suggest that MEK inhibition might represent a valid treatment option for patients with both mutated- or wild type-BRAF melanomas, affecting tumor growth through multiple targets.

Figure 1

Melanosphere isolation and validation. A) Image of melanospheres (left) and their differentiated progeny (right). B) Tumor volumes of xenografts generated by spheres or differentiated (diff) melanoma cells injected subcutaneously in Nude mice at the indicated cell doses. Mean ± SD of 3 independent experiments is shown. ** p < 0,01. C) Table of melanospheres tumorigenicity in dose response experiments. Cell numbers, number of mice injected and percentage of tumor engraftment is indicated for each condition. Tumors were monitored for 8 weeks post-injection. D) Hematoxylin and eosin (H&E) or immunohistochemistry for the indicated antigens performed on patient tumor or xenograft generated by melanospheres. The original magnification of each image is indicated.

Figure 2

Drug resistance of melanosphere and pathway activation. A) Cell viability of undifferentiated melanospheres of the indicated samples and melanocytes treated with the indicated drugs. Mean ± SD of 3 independent experiments is shown. ** p < 0,01. B) Cell viability of melanospheres (undifferentiated) and their progeny (differentiated) exposed to the indicated chemotherapeutic agents. A representative sample is shown. Mean ± SD of 3 independent experiments is shown. *** p < 0,001. C) Cell viability of melanospheres exposed to the indicated kinase inhibitors. Mean ± SD of 3 independent experiments is shown. ** p < 0,01; * p < 0,05 D) Immunoblot analysis of the indicated proteins or phosphoproteins in melanospheres. U251 and T98G glioblastoma cell lines were used as p-ERK positive and negative control, respectively.

Figure 3

Antitumor activity of PD0325901 on melanospheres and their progeny. A) Cell viability (Cell Titer Glo assay, Promega) of melanospheres with mutated- or wild type-BRAF treated with the indicated drug doses. Mean ± SD of 3 independent experiments is shown. *** p < 0,001. Cell cycle distribution (B) and immunoblot analysis of pathway activation (C) of melanospheres after a 2 day drug exposure. D) Percentage of AnnexinV positive cells in control or PD0325901-treated representative melanospheres samples with mutated- or wild type-BRAF. Mean ± SD of 3 independent experiments is shown. ** p < 0,01. E) Propidium Iodide staining and flow cytometric analysis of representative samples of melanospheres (stem) or differentiated (diff) melanoma cells with mutated- or wild type-BRAF untreated or exposed to PD0325901. The percentage of apoptotic cells with subdiployd DNA is indicated for each condition and cell type. Standard deviations of the percentages are indicated for each condition. ** ≤ 0,01, *** ≤ 0,001 compared to untreated controls.

Figure 4

Antitumor activity of PD in melanosphere-derived subcutaneous xenografts. Growth curves of xenografts derived from mutant-BRAF (A) or wild type-BRAF (C) melanospheres in control or PD0325901-treated mice. Mean ± SD of 3 independent experiments is shown. *** p < 0,001. B-D) Immunohistochemistry for KI-67, p-Erk and mouse CD34 in control or treated BRAF-mutated (B) or BRAF-wild type (D) xenografts. E) Immunoblot for VEGF expression in control or PD0325901-treated representative melanospheres with mutated- or wild type-BRAF. F) Immunohistochemistry for VEGF in control or PD0325901-treated xenografts.