Braf(V600E) cooperates with Pten loss to induce metastatic melanoma

Abstract

Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. To build a model of human melanoma, we generated mice with conditional melanocyte-specific expression of BRaf(V600E). Upon induction of BRaf(V600E) expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15-20 months. By contrast, expression of BRaf(V600E) combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma, indicating the presence of long-lived melanoma-initiating cells in this system. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanoma's cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.

Figure 1. Benign hyperplasias induced by melanocyte specific expression of BRaf^V600E

(a) Mice carrying various conditional alleles of BRaf (BRaf^CA) and/or Pten (Pten^lox4-5 or Pten^lox5) were crossed to Tyr::CreER mice with melanocyte specific expression of a hormone dependent form of Cre recombinase (CreER^T2) ,. 4-HT dependent activation of CreER leads to melanocyte specific conversion of BRaf^CA→BRaf^V600E and the conversion of the Pten^lox alleles to null alleles. BRaf^CA/+ (b), Tyr::CreER; BRaf^CA/+ (c) and Tyr::CreER; BRaf^CA/CA (d) mice were treated topically with 4-HT and monitored for 75 weeks for signs of melanocytic proliferation (i). Mice were euthanized and skin from the ear (ii, iii) and flank (iv) was stained with hematoxylin and eosin and inspected for the presence of pigmented cells. (e) A Tyr::CreER; BRaf^CA/+ mouse developed a papular pigmented lesion ∼60 weeks after topical administration of 4-HT (i). This mouse was euthanized and skin sections encompassing the lesion were stained with hematoxylin and eosin and inspected for the presence of pigmented cells (ii & iii).

Figure 2. BRaf^V600E cooperates with Pten loss in the induction of malignant melanoma

(a-c) Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mice were treated topically on the paw with 4-HT to elicit BRaf^V600E and to silence Pten expression. The presence of pigmented lesions was assessed 6 (a), 8 (b) and 10 (c) days following 4-HT administration. (d) Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} (i) were treated topically with 4-HT on the right ear. Mice were monitored for 7 weeks. Tyr::CreER; BRaf^CA/+; Pten^+/+ (e) and Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} (f) were treated topically with 4-HT on the right flank. Mice were euthanized at ∼7 weeks with the presence of malignant melanoma lesions assessed by visual inspection of the underside of the ventral/lateral skin. Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mice were treated topically on the ear, flank and tail with 4-HT. 25 days later mice were euthanized, skin sections were prepared, stained with hematoxylin and eosin and examined for the presence of pigmented cells. Almost confluent proliferation of densely pigmented cells was observed in the ear and skin (g) that penetrated deep into the dermis and the subcutis (h & i) and showed signs of pagetoid spread into the epidermis (h & k). Cells in these lesions stained positive with α-PEP1 antisera that detects expression of Tyrp1 (j & k). Pigmented cells in the lesions displayed histological characteristics of malignant melanoma, including marked cytological atypia, prominent nucleoli and aberrant mitotic figures (l) (j) Kaplan-Meier survival analysis of 4-HT treated Tyr::CreER; BRaf^CA/+; Pten^+/+ (n=22), Tyr::CreER; BRaf^+/+; Pten^{lox4-5/lox4-5} (n=5) and Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} (n=22) mice. Log rank tests of survival plots of the data indicated a statistically significant difference between the following survival curves: Tyr::CreER; BRaf^CA/+; Pten^+/+ versus Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} (p<0.0001) and; Tyr::CreER; BRaf^+/+; Pten^{lox4-5/lox4-5} versus Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} (p<0.0002)

Figure 3. BRaf^V600E cooperates with Pten loss in the induction of invasive and metastatic melanoma

(a) Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} mice were treated topically on the ear, flank and tail with 4-HT. 6 weeks later mice were euthanized and inspected for evidence of pigmented cells in the mammary gland lymph node. Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mice were treated similarly to those described in (a), euthanized and then inspected by microscopy at low (b) and high (c) power for the presence of pigmented cells in the lymph nodes. (d) A Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mouse was treated topically with 5mM 4-HT in 100%(v/v) ethanol on the distal tail. The mouse had a visible tumor within 4 weeks but did not require euthanasia until 24 weeks later after 4-HT administration. The presence of pigmented cells in the Iliac, lumbar, inguinal, axillary and submandibular lymph nodes was assessed by stereomicroscopy. Tumor cells were restricted to the iliac nodes (pictured). (e-f) Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} mice were treated topically on the ear, flank and tail with 4-HT. Mice were euthanized 6-7 weeks after 4-HT treatment at which times the lungs were excised, cleared of blood and visually inspected for the presence of pigmented lesions (e) and (f). Lung sections were prepared, stained with hematoxylin and eosin and examined for the presence of pigmented cells in the lung parenchyma (g).

Figure 4. Prevention of BRaf^V600E-induced melanomas by Rapamycin

(a) Adult Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mice were treated topically on the ear, flank and tail with 4-HT. The next day, mice were randomly assigned to be administered Rapamycin (7.5mg/kg, n=5) or solvent control (n=4) for 21 days. Mice were monitored daily for the presence of cutaneous malignant melanoma and euthanized according to a standard body conditioning score. After 3 weeks of Rapamycin treatment, drug administration was ceased and mice were monitored for the presence of cutaneous malignant melanoma as described above. Mouse survival was plotted using a Kaplan-Meier survival curve (c). (b) Representative images of a vehicle control (i) or a Rapamycin treated mouse (ii) immediately after the end of the 21 day drug administration are presented. (c) Kaplan-Meier survival curve of vehicle (n=4) or Rapamycin (n=5) treated cohorts of mice analyzed in this experiment. All mice were treated for the indicated 21 days as indicated in (a).

Figure 5. Prevention of BRaf^V600E-induced melanomas by PD325901

(a) Adult Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} mice were treated topically on the ear with 4-HT. One week later, mice were randomly assigned to be administered PD325901 (12.5mg/kg, n=12) or the solvent control (n=12) for 6 weeks. Mice were analyzed daily for the presence of cutaneous malignant melanoma and euthanized according to a standard body conditioning score. After 6 weeks of PD325901 treatment 7 drug treated and 6 control mice were euthanized for analysis of skin sections. A further 5 PD325901 treated and 6 control mice were monitored over the course of a further 11 weeks without any further drug administration. These mice were analyzed for the presence of cutaneous malignant melanoma as described above. Mouse survival was plotted using a Kaplan-Meier survival curve (c). (b) Representative anatomical and histological images of skin from 4-HT treated Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} mice analyzed after 6 weeks of treatment with control solvent (n=6, i) or PD325910 (n=7, ii) or from PD325901 treated mice left without further drug administration for an additional 9-11 weeks (iii). (c) Kaplan-Meier survival curve of vehicle (n=6) or PD325901 (n=5) treated cohorts of mice analyzed in this experiment. All mice were treated for the indicated 6 weeks as in (b). Log rank tests of survival plots of the data demonstrate a statistically significant difference between vehicle and PD325901 treated animals (p=0.0024). (d) 3 week old Tyr::CreER; BRaf^CA/+; Pten^{lox4-5/lox4-5} mice (n=10) were treated topically on the right ear with 4-HT. 10 days later all mice were administered PD325901 (12.5mg/kg) for 6 weeks. Drug administration was then ceased for 8 weeks. Mice were then randomized into two groups: Group A (blue) was administered vehicle control and Group B (red) was administered PD325901 for a further 6 weeks. At the end of this period mice were monitored prospectively for disease progression as described above. Mouse survival was plotted using a Kaplan-Meier survival curve that demonstrated statistically significant difference (p=0.0018) between the groups by log rank tests (e).

Figure 6. Melanoma regression in response to combination treatment with PD325901 and Rapamycin

(a) Melanoma was initiated in a group of 20 Tyr::Cre; BRaf^CA; Pten^lox/lox mice by local administration of 1-2μl of 5mM 4-HT to back skin. 3 weeks later, when the animals had readily measurable melanoma lesions, mice were randomly divided into 4 groups that were administered either: 1. Solvent control (Vehicle, blue); 2. PD325901 (PD, 12.5mg/kg, red); 3. Rapamycin (Rapa, 7.5mg/kg, green) or; 4. The combination of both Rapamycin and PD325901 (PD+Rapa, orange) for 3 weeks as indicated. (b) Tumor size in each of the treatment groups described in (a) was measured daily and plotted as percentage change in tumor size compared to the starting size. Two-way ANOVA analysis demonstrates statistical significance: 1. Vehicle vs Rapa or PD or PD+Rapa (p<0.0001); 2. PD vs PD+Rapa (p=0.0024) and; 3. Rapa vs PD+Rapa (p=0.0120). (c) 2 hours following the final administration of the various agents, mice were euthanized and melanoma specimens were prepared for staining with H&E or antisera against Ki67, pERK1/2 or p4EBP1 as indicated.

Figure 7. Inhibition of mouse melanoma cell proliferation by Rapamycin or PD325901 in vitro

A melanoma cell line (2697T) was derived from a 4-HT induced lesion that developed on the skin of a white Tyr::CreER; BRaf^CA/+; Pten^lox5/lox5 mouse. 2697T cells growing asynchronously in full media were treated with PD325901 (2μM) or Rapamycin (50nM) as indicated. (a) Cell growth was measured using a standard Crystal Violet staining assay. (b) DNA synthesis and content was assessed at 48 hours using standard anti-BrdU/Propidium Iodide FACS analysis following a 2 hour BrdU pulse. The percentage of cells in each portion of the cell cycle is indicated. (c) 2697T cells, growing asynchronously in full media, were treated with 2μM PD325901 for 24-72 hours at which time cell extracts were prepared. Expression of Bim-EL, ERK1/2, Akt1, Caspase 3 and its active cleavage product (arrowhead) as well as the phosphorylation of ERK1/2 and Akt1 was assessed by immunoblotting. (d) Serum deprived 2697T cells were pre-treated with different concentrations of Rapamycin (2.5-250nM) for 30 minutes prior to stimulation with 10%(v/v) fetal calf serum for a further 20 minutes. Expression of Akt1, MEK1/2 and Actin as well as the phosphorylation of 4EBP1, p70^S6K, and Akt1 (S473 and T308) was assessed by immunoblotting.