Inhibition of Mammalian target of rapamycin by rapamycin causes the regression of carcinogen-induced skin tumor lesions

Abstract

Purpose: The activation of Akt/mammalian target of rapamycin (mTOR) pathway represents a frequent event in squamous cell carcinoma (SCC) progression, thus raising the possibility of using specific mTOR inhibitors for the treatment of SCC patients. In this regard, blockade of mTOR with rapamycin prevents the growth of human head and neck SCC cells when xenotransplanted into immunodeficient mice. However, therapeutic responses in xenograft tumors are not always predictive of clinical anticancer activity.

Experimental design: As genetically defined and chemically induced animal cancer models often reflect better the complexity of the clinical setting, we used here a two-step chemical carcinogenesis model to explore the effectiveness of rapamycin for the treatment of skin SCC.

Results: Rapamycin exerted a remarkable anticancer activity in this chemically induced cancer model, decreasing the tumor burden of mice harboring early and advanced tumor lesions, and even recurrent skin SCCs. Immunohistochemical studies on tumor biopsies and clustering analysis revealed that rapamycin causes the rapid decrease in the phosphorylation status of mTOR targets followed by the apoptotic death of cancer cells and the reduction in the growth and metabolic activity of the surviving ones, concomitant with a decrease in the population of cancer cells expressing mutant p53. This approach enabled investigating the relationship among molecular changes caused by mTOR inhibition, thus helping identify relevant biomarkers for monitoring the effectiveness of mTOR inhibition in the clinical setting.

Conclusions: Together, these findings provide a strong rationale for the early evaluation of mTOR inhibitors as a molecular targeted approach to treat SCC.

Figure 1

Anti-tumoral effects of rapamycin in early lesions of chemically-induced skin tumors. Mice were treated with a single dose of the carcinogen DMBA (initiation), followed by the twice weekly treatment with TPA (promotion). The number and diameter of each tumor was measured weekly, and plotted as a function of time (weeks) after the exposure to DMBA. Rapamycin (RP; 10 mg/kg) or vehicle (V) treatment started at week 9. The second phase of the rapamycin treatment in the rapamycin-treated group was reinitiated at week 24, as indicated. Bar, number of tumors of each size (indicated by color coding). Arrows, rapamycin treatment. X, week in which mice had to be sacrificed due to their tumor burden. Representative IF analysis of the tissues from the corresponding vehicle and rapamycin treated animals. The phosphoS6 (pS6; in red) immunodetection reflected the mTOR status and E-cadherin (in green) was used to label tumor cells. Cell nuclei were stained with DAPI (in blue).

Figure 2

Antitumoral effect of rapamycin in advanced skin cancer lesions. The number and size of chemically-induced tumors in mice treated with vehicle (V) and rapamycin (RP; 10 mg/kg) was depicted as for Figure 1. Bar, number of tumors of the indicated size based on their designated color coding. Rapamycin treatment was initiated when most of the animals had at least 1-2 large tumors, as indicated. The control group had to be sacrificed 6 weeks after treatment initiation with vehicle, as indicated with an X. Arrows, (RP) rapamycin or (V) vehicle treatment. Representative tumors were photographed before rapamycin initiation (Pre-treatment) and after 6 weeks of the initial treatment with rapamycin or vehicle control.

Figure 3

Immunohistochemical analysis of biomarkers of rapamycin treatment response. A. IHC from representative tumors acquired from control and rapamycin-treated mice 7 days after initiating the rapamycin treatment as described in Figure 2. The antibodies used are indicated. B. Hierarchical clustering of IHC staining score against different biomarkers from the control tumors (C) and the papillomas harvested at different time points after rapamycin administration (R1, day 1; R3, day 3; R5, day 5; and R7, day 7). Deep red, high staining scores (strong staining intensity and 75-100% of positive staining cells). Yellow, low staining scores (weak staining intensity and less than 10% of cells positive for IHC). C. TUNEL staining showing the apoptotic cells (in red) of tumor sections from the control and rapamycin-treated mice after 3 days of the rapamycin treatment initiation. Cell nuclei were stained with DAPI (in blue).