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. 2014 Nov 20;33(47):5442-9.
doi: 10.1038/onc.2013.489. Epub 2013 Nov 18.

Transformation by Hras(G12V) is consistently associated with mutant allele copy gains and is reversed by farnesyl transferase inhibition

X Chen  1 , J M Makarewicz  1 , J A Knauf  2 , L K Johnson  3 , J A Fagin  4
Affiliations

Transformation by Hras(G12V) is consistently associated with mutant allele copy gains and is reversed by farnesyl transferase inhibition

X Chen et al. Oncogene. .

Abstract

RAS-driven malignancies remain a major therapeutic challenge. The two-stage 7,12-dimethylbenz(a)anthracene (DMBA)/12-o-tetradecanoylphorbol-13-acetate (TPA) model of mouse skin carcinogenesis has been used to study mechanisms of epithelial tumor development by oncogenic Hras. We used mice with an Hras(G12V) knock-in allele to elucidate the early events after Hras activation, and to evaluate the therapeutic effectiveness of farnesyltransferase inhibition (FTI). Treatment of Caggs-Cre/FR-Hras(G12V) mice with TPA alone was sufficient to trigger papilloma development with a shorter latency and an ∼10-fold greater tumor burden than DMBA/TPA-treated WT-controls. Hras(G12V) allele copy number was increased in all papillomas induced by TPA. DMBA/TPA treatment of Hras(G12V) knock-in mice induced an even greater incidence of papillomas, which either harbored Hras(G12V) amplification or developed an Hras(Q61L) mutation in the second allele. Laser-capture microdissection of normal skin, hyperplastic skin and papillomas showed that amplification occurred only at the papilloma stage. HRAS-mutant allelic imbalance was also observed in human cancer cell lines, consistent with a requirement for augmented oncogenic HRAS signaling for tumor development. The FTI SCH66336 blocks HRAS farnesylation and delocalizes it from the plasma membrane. NRAS and KRAS are not affected as they are alternatively prenylated. When tested in lines harboring HRAS, NRAS or KRAS mutations, SCH66336 delocalized, inhibited signaling and preferentially inhibited growth only of HRAS-mutant lines. Treatment with SCH66336 also induced near-complete regression of papillomas of TPA-treated Hras(G12V) knock-in mice. These data suggest that farnesyl transferase inhibitors should be reevaluated as targeted agents for human HRAS-driven cancers, such as those of bladder, thyroid and other epithelial lineages.

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Figures

Fig. 1
Fig. 1. Topical administration of TPA to HrasG12V mice triggers papilloma development
Average number of tumors per mouse during the time course of the study. There were five groups of mice: HrasG12V mice treated with a single dose of DMBA followed by TPA treatment twice per week (n=7), Hras G12V mice treated with TPA only (n=7), wild type mice treated DMBA followed by TPA (n=7), Hras G12V mice treated with the vehicle acetone (twice per week) (n=8) and FR-Hras mice (harboring the targeted Hras allele, unrecombined, because of the absence of Caggs-Cre) treated with TPA (n=7).
Fig. 2
Fig. 2. Hras allelic imbalance in papillomas from HrasG12V mice
A) PCR of genomic DNA of 10 papillomas or tails from TPA-treated HrasG12V mice with primers that distinguish mutant from WT Hras alleles. W: wild-type 622 bp allele; M: 666bp targeted allele. The mutant Hras allele copy number was increased in all 10 papillomas induced by TPA alone. B) PCR of DNA from 10 randomly selected papillomas from DMBA/TPA-treated HrasG12V mice, tails from HrasG12V mice and papillomas from DMBA/TPA treated WT mice. 7/10 papillomas had M>W allelic imbalance. The three lanes marked by an asterisk had ~ 1/1 ratio of M/W alleles. C) Representative Hras Sanger sequence traces of PCR products of tumor DNAs from panel B: Left: Tumor with M>W Hras allelic imbalance, showing the Hras G12V mutation, and a WT Hras Q61 codon. Right: Tumor (*) without Hras allelic imbalance (M=W), harboring both Hras G12V and Q61L mutations. G12V and Q61L are located in different alleles (Suppl Fig 2). D) Laser-capture microdissection (LCM) of normal skin epithelium, hyperplasia and papilloma. PCR of genomic DNA of LCM of tissues from TPA-treated HrasG12V mice. As indicated, Hras mutant allele amplification occurred only at the papilloma stage. N: normal skin epithelium; H: hyperplasia; P: papilloma.
Fig. 3
Fig. 3. Growth of human cancer cell lines with HRAS mutation is preferentially inhibited by SCH 66336
A) IC50 of each cell line for SCH 66336. The indicated cell lines were incubated with different concentrations of SCH 66336 for 4 days and the cell number counted. All 5 HRAS mutant cell lines have lower IC50 for FTI compared to cells with NRAS or KRAS mutation. B) Growth of cell lines in the presence of SCH 66336. The indicated cell lines were incubated with SCH 66336 (25nM, 100nM) or vehicle for 2, 4 and 6 days. C) Left panel: FACS for FITC-labeled BrdU vs 7-AAD for representative HRAS (SKmel31) and NRAS (Hth7) mutant cell lines treated for 3 days with 100 nM SCH 66336. Right panel: SCH 66336 decreased DNA synthesis in the 4 HRAS mutant but not in NRAS or KRAS mutant cell lines.
Fig. 4
Fig. 4. SCH 66336 disrupts HRAS membrane localization and inhibits MAPK signaling in HRAS mutant lines
A) Western blots of subcellular fractions of Hth83 cells treated with 250nM SCH 66336 for 48hr, probed with the indicated antibodies. SCH 66336 delocalized HRAS, but not KRAS or NRAS. C: Cytoplasmic; M: Membrane; N: Nucleus. B) SCH 66336 inhibits HRAS signaling with a delayed time course in Hth83 cells. Hth83 cells were incubated with 250nM SCH 66336 for the indicated time. The cell lysates were Western blotted with the indicated antibodies. U: unfarnesylated HRAS; F: farnesylated HRAS. C) SCH 66336 blocked MAPK signaling in a concentration-dependent manner. Western blots of 4 HRAS mutant cell lines treated with the indicated concentration of SCH 66336 for 72hr probed with indicated antibodies. D) SCH 66336 had no effect on MAPK signaling in cell lines with KRAS or NRAS mutations, despite inhibiting farnesylation of WT HRAS in these lines.
Fig. 5
Fig. 5. SCH 66336 induces regression of papillomas in HrasG12V mice
A) Representative photos of mice after treatment with vehicle or SCH 66336 for the indicated times. HrasG12Vmice were first treated with TPA for 12 to 16 weeks to induce papilloma development. Mice then were treated with 80mg/kg SCH 66336 b.i.d. (n=5) or vehicle (n=6) by gavage for the indicated times. B) Tumor volumes at the indicated times during treatment with vehicle (n=10) or SCH 66336 (n=11) from the mice described above. Tumor size was measured on days 0, 3, 5, 7 and 10. Bars represent mean ± SEM. C) SCH 66336 decreased cell proliferation and pERK in papillomas. Representative H&E and IHC staining for Ki67 and pERK in sections of mouse papillomas treated with vehicle or SCH 66336. D) Western blot of liver tissues from HrasG12Vmice treated with vehicle (n=4) or SCH 66336 (n=4), probed with the indicated antibodies. SCH 66336 decreased MAPK signaling and inhibited Hras farnesylation in livers of HrasG12V mice.
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