Exploratory comparisons between different anti-mitotics in clinically-used drug combination in triple negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) constitutes a very aggressive type of breast cancer with few options of cytotoxic chemotherapy available for them. A chemotherapy regimen comprising of doxorubicin hydrochloride and cyclophosphamide, followed by paclitaxel, known as AC-T, is approved for usage as an adjuvant treatment for this type of breast cancer. In this study we aimed to elucidate the role of KIF11 in TNBC progression throughout its inhibition by two synthetic small molecules containing the DHPM core (dihydropyrimidin-2(1H)-ones or -thiones), with the hypothesis that these inhibitors could be an interesting option of antimitotic drug used alone or as adjuvant therapy in association with AC. For this purpose, we evaluated the efficacy of DHPMs used as monotherapy or in combination with doxorubicin and cyclophosphamide, in Balbc-nude mice bearing breast cancer induced by MDA-MB-231, having AC-T as positive control. Our data provide extensive evidence to demonstrate that KIF11 inhibitors showed pronounced antitumor activity, acting in key points of tumorigenesis and cancer progression in in vivo xenograft model of triple negative breast cancer, like down-regulation of KIF11 and ALDH1-A1. Moreover, they didn't show the classic peripheral neuropathy characterized by impaired mobility, as it is common with paclitaxel use. These results suggest that the use of a MAP inhibitor in breast cancer regimen treatment could be a promising strategy to keep antitumoral activity reducing the side effects.

Keywords: KIF11 inhibition; Kinesin Eg5; adjuvant treatment; breast cancer; cancer progression.

Figure 1. Body weight and tumor volume monitoring during monotherapy and drug combination therapy.

(A) Body weight of animals submitted to monotherapy, administered with drug diluent (vehicle group), 4bt (50 or 80 mg/Kg), 4bc (50 or 80 mg/Kg), 4bt + 4bc (25 mg/Kg each) or paclitaxel (20 mg/Kg) or (B) to drug combination, doxorubicin (10 mg/Kg) + cyclophosphamide (100 mg/Kg) in a single dose, followed by three doses of KIF11 inhibitor 4bt (80 mg/Kg) or paclitaxel (10 mg/Kg) was monitored twice a week. Tumor volume for monotherapy (C) and drug combination (D) was also checked during all treatment time. (E) An in vitro survival assay with MDA-MB-231 cells was performed to evaluate if the association of paclitaxel or 4bt could potentiate antitumor effect of drug combination (doxorubicin and cyclophosphamide) treatment. (F) A representative image of tumor for each experimental group. Lines and columns represent mean ± SEM of five animals used in each experimental group. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 as compared to control group; ^# p < 0.05 compared to AC treatment group.

Figure 2. Histopathological analysis of tumor sections upon monotherapy or drug combination treatments.

Representative images of paraffin-embedded sections of tumors stained with H&E from the different experimental groups administered intraperitoneally (i.p.) with: (A) Vehicle, (B) 4bt (80 mg/Kg), (C) 4bc (80 mg/Kg), (D) paclitaxel (20 mg/Kg), (E) Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by paclitaxel (10 mg/Kg) (AC-T) and (F) Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by KIF11 inhibitor 4bt (AC-4bt). Treatment with 4bt used as monotherapy or in drug combination prevent exacerbated tumor growth leading to a decrease in the extension of the central necrotic area (outlined and marked with asterisk). Bars: 250 μm (A, D, E and F) and 200 μm (B and C).

Figure 3. Analysis of intratumoral KIF11 expression.

(A) Tumor sections from the different experimental groups administered intraperitoneally (i.p.) with: Vehicle, 4bt (80 mg/Kg), 4bc (80 mg/Kg), paclitaxel (20 mg/Kg), Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by paclitaxel (10 mg/Kg) (AC-T) and Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by KIF11 inhibitor 4bt (AC-4bt) were immunostained for KIF11 (green) and genetic material (blue). (B) Integrated Density calculated by using sets containing 10 random images of each experimental group. 4bt or drug combinations were able to significantly decrease intratumoral KIF11 expression levels. Data represent mean ± SEM of three independent experiments. ^* p < 0.05, ^**** p < 0.0001 as compared to control group (vehicle). Bars: 25 μm.

Figure 4. Proliferation analysis of tumor sections.

(A) Tumor sections from the different experimental groups administered intraperitoneally (i.p.) with: Vehicle, 4bt (80 mg/Kg), 4bc (80 mg/Kg), paclitaxel (20 mg/Kg), Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by paclitaxel (10 mg/Kg) (AC-T) and Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by KIF11 inhibitor 4bt (AC-4bt) were immunostained for Ki67 (green) and genetic material (blue). (B) Percentage of Ki67 positive nuclei for each experimental group. Data represent mean ± SEM of three independent experiments. ^** p < 0.01, ^**** p < 0.0001 as compared to control group (vehicle). Bars: 25 μm.

Figure 5. TUNEL analysis for apoptosis detection in tumor sections of xenografted Balb-c/nude mice.

(A) Representative images of TUNEL assay of tumor sections from the different experimental groups administered intraperitoneally (i.p.) with: Vehicle, 4bt (80 mg/Kg), 4bc (80 mg/Kg), paclitaxel (20 mg/Kg), Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by paclitaxel (10 mg/Kg) (AC-T) and Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by KIF11 inhibitor 4bt (AC-4bt). (B) Quantitative analyses of apoptotic cell area for each experimental group. Data represent mean ± SEM of three independent experiments. ^*** p < 0.001, ^**** p < 0.0001 as compared to control group (vehicle). Bars: 50 μm.

Figure 6. ALDH1-A1 expression analysis in tumor sections of xenografted Balb-c/nude mice.

(A) Representative images of ALDH1-A1 immunostaining of tumor sections from the different experimental groups administered intraperitoneally (i.p.) with: Vehicle, 4bt (80 mg/Kg), 4bc (80 mg/Kg), paclitaxel (20 mg/Kg), Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by paclitaxel (10 mg/Kg) (AC-T) and Doxorubicin (10 mg/Kg) + Cyclophosphamide (100 mg/Kg) followed by KIF11 inhibitor 4bt (AC-4bt). (B) Quantitative analyses highly positive and positive area with indication of the score for each experimental group. L-POS: low positive; POS: positive. Data represent mean ± SEM of three independent experiments. ^*** p < 0.001, ^**** p < 0.0001 as compared to control group (vehicle). Bars: 50 μm.

Figure 7. Scheme of therapeutic regimen applied in Balb-c/nude mice bearing breast cancer.

(A) For monotherapy, animals were administered i.p. with six doses of 4bt, 4bc (50 or 80 mg/Kg), 4bt + 4bc (25 mg/Kg each) or paclitaxel (20 mg/Kg) twice a week. (B) For drug combination, animals received one dose of doxorubicin (10 mg/Kg) + cyclophosphamide (100 mg/Kg) i.p. and after 1.5 weeks three doses of 4bt (80 mg/Kg) or paclitaxel (10 mg/Kg) were administered twice a week.