Insulin-like growth factors I and II receptors in the breast cancer survival disparity among African-American women

Abstract

Objective: African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) women. We have recently shown higher insulin-like growth factor II (IGF-II) expression in paired breast tissue samples from AA women as compared to CA women. IGF-II is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I and Insulin receptors (IGF-IR, IR) while IGF-II circulating levels are regulated by cellular uptake through the IGF2 receptor. We hypothesize that differential expression of the IGF1R and IGF2R among AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups.

Design: We examined IGF-IR and IGF2R mRNA, protein expression and IGF1R phosphorylation in paired breast tissue samples from AA and CA women by Real Time-PCR, Western blot analysis, immunohistochemistry and ELISA techniques.

Results: Our results showed significantly increased expression of IGF1R in AA normal tissues as compared to CA normal tissues. IGF1R expression was similar between AA normal and malignant tissues, while IGF1R, IRS-1 and Shc phosphorylation was significantly higher in AA tumor samples. Significantly higher levels of IGF2R were found in CA tumor samples as compared to AA tumor samples.

Conclusions: We conclude that IGF1R and IGF2R differential expression may contribute to the increased risk of malignant transformation in young AA women and to the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets in breast cancer.

Figure 1

IGF-1R gene expression in African-American and Caucasian paired breast tissue samples assessed by Real Time-PCR (RT-PCR). Figure 1(A–B) shows IGF-1R gene expression represented as fold change after normalization using GAPDH as an internal control. Two (2) or more fold change was considered significant (*). Figure 1A shows IGF-1R mRNA fold change between AAM and CAM samples divided into pre-menopausal and post-menopausal women. Figure 1B shows IGF-1R mRNA expression shown as fold change between AAN and CAN. AAN=African-American normal tissue, AAM=African-American malignant tissue, CAN=Caucasian normal tissue and CAM=Caucasian malignant tissue. Total number of patients (n) analyzed per group was as follows: AAN= 23, AAM=27, CAN=20 and CAM=24.

Figure 2

IGF2R gene expression in African-American and Caucasian paired breast tissue samples assessed by Real Time-PCR (RT-PCR). Figure 2(A–B) shows IGF2R gene expression represented as fold change after normalization using GAPDH as an internal control. Two (2) or more fold change was considered significant (*). Figure 2A shows IGF2R mRNA fold change between AAM and CAM samples (pre-menopausal and post-menopausal women). Figure 2B shows IGF2R mRNA comparison between AAN and CAN. AAN=African-American normal tissue, AAM=African-American malignant tissue, CAN=Caucasian normal tissue and CAM=Caucasian malignant tissue. Total number of patients (n) analyzed per group was as follows: AAN= 23, AAM=27, CAN=20 and CAM=24.

Figure 3

Representative Western blot analyses of IGF-1R in paired tissue samples from African-American (AA) and Caucasian (CA) women. The samples were separated into pre-menopausal (figure 3A) and post-menopausal (figure 3B) groups based on the higher incidence of breast cancer in AA women younger than 45 years (but not after 45 years). Immunoreactive bands for IGF-1R and cytokeratin 18 were identified using ECL, scanned by densitometry and normalized to cytokeratin 18. The 130 kDa band represents mature IGF-1R. Cytokeratin 18 was used as an epithelial cell marker (45 kDa). Lower panel (3C) show bar graphs of IGF-1R data normalized to cytokeratin 18 and presented as the mean ± SE of all samples per group. Asterisks indicate values statistically different (*p<0.05). Total number of patients (n) analyzed per group was as follows: AAN= 23, AAM=27, CAN=20 and CAM=24.

Figure 4

Representative Western blot analyses of IGF2R in paired tissue samples from African-American (AA) and Caucasian (CA) women. The samples were separated into pre-menopausal (figure 4A) and post-menopausal (figure 4B) groups. Immunoreactive bands for IGF-1R and cytokeratin 18 were identified using ECL, scanned by densitometry and normalized to cytokeratin 18. The 270 kDa band represents pro-IGF2R. Cytokeratin 18 was used as an epithelial cell marker (45 kDa). Lower panel (4C) show bar graphs of IGF2R data normalized to cytokeratin 18 and presented as the mean ± SE of all samples per group. Asterisks indicate values statistically different (*p<0.05). Total number of patients (n) analyzed per group was as follows: AAN= 23, AAM=27, CAN=20 and CAM=24.

Figure 5

IGF-1R phosphorylation study using the PathScan^® phospho-IGF1 receptor β (tyr 1131) ELISA kit. Figure 5A shows a bar graph representation of IGF-1R phosphorylation measured as OD 450 for all samples per group (AAN= 23, AAM=27, CAN=20 and CAM=24). Asterisks indicate values statistically different (*p<0.05). Figure 5B shows representative western blot analyses of total and phosphorylated IGF1R, IRS-1 and Shc proteins. Figure 5C–D show bar graphs of phosphorylated IRS-1 and Shc normalized to total IRS-1 and Shc proteins and presented as the mean ± SE of all samples per group. Asterisks indicate values statistically different (*p< 0.05).

Figure 5

IGF-1R phosphorylation study using the PathScan^® phospho-IGF1 receptor β (tyr 1131) ELISA kit. Figure 5A shows a bar graph representation of IGF-1R phosphorylation measured as OD 450 for all samples per group (AAN= 23, AAM=27, CAN=20 and CAM=24). Asterisks indicate values statistically different (*p<0.05). Figure 5B shows representative western blot analyses of total and phosphorylated IGF1R, IRS-1 and Shc proteins. Figure 5C–D show bar graphs of phosphorylated IRS-1 and Shc normalized to total IRS-1 and Shc proteins and presented as the mean ± SE of all samples per group. Asterisks indicate values statistically different (*p< 0.05).

Figure 6

Immunohistochemistry of IGF-1R and IGF2R in human normal and malignant breast tissue samples. Malignant AA and CA samples correspond to ER (+) invasive ductal carcinomas (IDC) Bloom and Richardson’s grade III. Panels A-D corresponds to IGF-1R and IGF2R immunostaining from AAN samples. Panels E-H corresponds to IGF-1R and IGF2R immunostaining from AAM samples. Panels I-L and M-P correspond to IGF-1R and IGF2R immunostaining, in CAN and CAM samples respectively. Original magnifications 20X (insert represent a 40X section). Total number of patients (n) analyzed per group was as follows: AAN= 15, AAM=15, CAN=13 and CAM=13.