Caloric restriction augments radiation efficacy in breast cancer

Abstract

Dietary modification such as caloric restriction (CR) has been shown to decrease tumor initiation and progression. We sought to determine if nutrient restriction could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (IR) and alter the molecular profile of triple-negative breast cancer (TNBC), which displays a poor prognosis. In two murine models of TNBC, significant tumor regression is noted with IR or diet modification, and a greater regression is observed combining diet modification with IR. Two methods of diet modification were compared, and it was found that a daily 30% reduction in total calories provided more significant tumor regression than alternate day feeding. At the molecular level, tumors treated with CR and IR showed less proliferation and more apoptosis. cDNA array analysis demonstrated the IGF-1R pathway plays a key role in achieving this physiologic response, and multiple members of the IGF-1R pathway including IGF-1R, IRS, PIK3ca and mTOR were found to be downregulated. The innovative use of CR as a novel therapeutic option has the potential to change the biology of tumors and enhance the opportunity for clinical benefit in the treatment of patients with TNBC.

Keywords: IGF; breast cancer; caloric restriction; cytotoxic therapy; radiation; tumor regression.

Figure 1. Experimental design. Tumors were generated by injecting either 67NR or 4T1 cells into the mammary fat pad of balb/c mice. Once tumors were palpable, mice were treated with IR to the primary tumor alone, treated with a dietary intervention of either ADF or CR or treated with a combination of the two and compared with control mice given no IR and an ad libitum diet.

Figure 2. Tumor re-growth-delay curves for tumors created from two TNBC cell lines: 4T1 and 67NR cells using two different methods of caloric restriction. Mice were treated with each of the following conditions for 67NR (A) cell lines: ad libitum (AL) diet, radiation (RT), alternate day feeding (ADF) or ADF + RT, and their weights were recorded (B). From the reference of the AL cohort for 67NR tumors at 1,000 mm³, RT gave a 16% growth delay, ADF 14%, ADF + RT 25%. From the reference of the AL cohort for 4T1 and weights were tumors at 1,000 mm³ (C), RT gave a 23% growth delay, ADF 30%, ADF + RT 45% also recorded for this cohort (D). Using the 4T1 tumors, the ADF method was then compared with a 30% reduction in calories (CR) (E) using: AL, RT, CR and CR + RT. From the reference of the AL cohort for 4T1 tumors at 1,000 mm³, RT gave a 23% growth delay, CR 56%, CR+RT 82% with an average weight reduction (F) of 12%. Based on this, the optimal method for performing future studies would be a constant reduction of 30% of the caloric intake. *Denotes p < 0.05 by Student’s t-test at 1,000 mm³.

Figure 3. Molecular effects of treatment. Proliferation was evaluated by Ki-67 (A), and bcl-2 was used to evaluate apoptosis (B) for all conditions: Ad lib, CR, IR and CR + IR. Combination therapy was noted to decrease proliferation and increase apoptosis in tumors. qRT-PCR of IGF-1R pathway molecules shows downregulation (C) with either CR, IR or both. *Notes statistical significance with a p < 0.05.

Figure 4. Immunohistochemical investigation of proliferation and IGF-1R pathway. Proliferation was also evaluated by immunohistochemistry (IHC) with Ki-67 staining, which was noted to have higher proliferation in the ad libitum tumors (A) compared with the tumors treated with CR and IR. IGF-1R was noted to have positive staining (3+) in AL tumors and negative (1+) in CR + IR samples while the downstream target of GSK-3B revealed positive staining (3+) in AL tumors and 2+ staining in CR + IR tumors.