Chemical carcinogen-induced rat mammary carcinogenesis is a potential model of p21-activated kinase positive female breast cancer

Abstract

The p21-activated kinase 1 (PAK1) gene encodes a serine/threonine kinase that is overexpressed in a subset of human breast carcinomas with poor prognosis. The laboratory rat (Rattus norvegicus) orthologous gene is located at Mammary carcinoma susceptibility 3 (Mcs3) QTL on rat chromosome 1. We used quantitative PCR to determine effects of Mcs3 genotype and 7,12-dimethylbenz(a)anthracene (DMBA) exposure on Pak1 expression. There was no effect of Mcs3 genotype; however, there was a 3.5-fold higher Pak1 level in DMBA-exposed mammary glands (MGs) than in unexposed glands (P < 0.05). Sequence variants in Pak1 exons did not alter amino acid sequence between Mcs3-susceptible and -resistant strains. Protein expression of PAK1/Pak1 in human breast carcinomas and DMBA-exposed rat mammary glands was detected using immunohistochemistry (IHC). Rat mammary glands from 12-wk-old females unexposed to DMBA were negative for Pak1, whereas 24% of carcinogen-exposed mammary glands from age-matched females stained positive for Pak1. The positive mammary glands exposed to carcinogen had no pathological signs of disease. Human breast carcinomas, used as comparative controls, had a 22% positivity rats. This was consistent with other human breast cancer studies of PAK1 expression. Similar frequencies of human/rat PAK1/Pak1 expression in female breast carcinomas and carcinogen-induced rat mammary glands, showing no visible pathogenesis of disease, suggests aberrant PAK1 expression is an early event in development of some breast cancers. Laboratory rats will be a useful experimental organism for comparative studies of Pak1-mediated mechanisms of breast carcinogenesis. Future studies of PAK1 as a diagnostic marker of early breast disease are warranted.

Keywords: animal models of breast cancer; breast cancer; ductal carcinoma in situ; p21-activated kinase; rat Mammary Carcinoma Susceptibility 3 (Mcs3).

Figure 1.

Increased Pak1 transcript levels in DMBA-induced rat mammary glands. RT-qPCR of Pak1 and Ilk levels in rat mammary gland tissue 4 wk after DMBA-induction of mammary carcinogenesis compared with age-matched uninduced controls. qPCRs, run in triplicate for each sample, were used to measure target gene quantity standardized by Rplp2 quantity. QPCR Ct values were quantified using linear standard curves derived from known cDNA concentrations. P values are from two-way ANOVA F tests including exposure and genotype. Mcs3 genotypes were pooled because genotype was not significant for either Pak1 or Ilk. (A: n = 21 DMBA rats, 21 control rats; P = 0.02. B: n = 21 DMBA rats, 21 control rats; P = 0.47). ANOVA, analysis of variance; cDNA, complementary DNA; Ct, threshold cycle; DMBA, 7,12-dimethylbenz(a)anthracene; Ilk, Integrin-linked kinase; Mcs3, Mammary carcinoma susceptibility 3; Pak1, p21-activated kinases; qPCR, quantitative polymerase reaction; RT-qPCR, quantitative reverse transcription PCR.

Figure 2.

Correlation of Pak1 and Ilk expression in rat mammary glands is lost with DMBA exposure. Standardized values of Pak1 and Ilk expression from previous RT-qPCR of rat mammary gland tissue (Fig. 1) were analyzed by Pearson’s correlation. A: rat strain E (susceptible) mammary glands, unexposed to DMBA (n = 12 control rats, r = 0.1811, P = 0.5732). B: strain E age-matched rat mammary glands 4 wk after DMBA-induction of mammary carcinogenesis (n = 10 DMBA rats, r = −0.2158, P = 0.5494). C: strain D (resistant) rat mammary glands, unexposed to DMBA (n = 8 control rats, r = 0.9349, P = 0.0007). D: strain D age- matched rat mammary glands, 4 wk after DMBA-induction of mammary carcinogenesis (n = 10 DMBA rats, r = 0.5275, P = 0.1171). DMBA, 7,12-dimethylbenz(a)anthracene; Ilk, Integrin-linked kinase; Pak1, p21-activated kinases.

Figure 3.

Representative Pak1 staining images of morphologically normal DMBA-induced and uninduced rat mammary glands. IHC images of mammary ducts and smaller ductules of a morphologically normal rat mammary gland, 4 wk after DMBA-induction of mammary carcinogenesis (left, two columns) compared with an age-matched rat, unexposed to DMBA (right, two columns). Darker staining, relative to the IgG control, is evident in the DMBA-exposed mammary ducts but not in the uninduced control mammary gland. H&E staining is shown for a histological view of tissue sections. Images were taken at ×40 magnification on an Aperio ImageScope CS2. DMBA, 7,12-dimethylbenz(a)anthracene; IgG, immunoglobulin G; IHC, immunohistochemistry; H&E, hematoxylin and eosin; Pak1, p21-activated kinases.

Figure 4.

Images of PAK1-positive staining in human breast tissue. Representative IHC images from ER+/HER2− ductal carcinoma in situ (DCIS) (A), ER+/HER2− invasive ductal carcinoma (IDC) (B), HER2 + IDC (C), and triple-negative IDC (D). Darker PAK1 staining is evident compared with respective IgG controls. H&E staining is shown to provide a detailed view of the tissue. Images were taken at ×40 magnification on an Aperio ImageScope CS2. DCIS, ductal carcinoma in situ; H&E, hematoxylin and eosin; IDC, invasive ductal carcinoma; IgG, immunoglobulin G; IHC, immunohistochemistry; PAK1, p21-activated kinases.