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.

INTRODUCTION

Amplification of human chromosome 11q13/14 in breast cancer is associated with poor prognosis, with patient tumors displaying chemoresistance and a 10-year survival rate of only 50% (1–5). Amplification of 11q13/14 contains several known and putative driver genes, but it remains unclear if a cancer phenotype is driven by a cassette of genes, or a single oncogene (2, 6). A recent study which profiled nearly 2,500 primary breast tumors with copy number aberrations determined 11q13/14 contains two gene cassettes, one centered around cyclin D1 (CCND1, 11q13.3), and the other around p21-activated kinase (PAK1, 11q14.1) (4).

Human breast epithelial cells normally have low PAK1 expression; however, amplification of PAK1 is found in 33% of breast tumors and cancer cell lines (7–10). PAK1 gene expression is associated with aggressive breast cancer type, histological grade, and proliferation level (7, 8, 11). A meta-analysis of 15 independent studies that investigated association of PAK1 with disease prognosis, found that high PAK1 expression in tumors is negatively associated with overall survival and disease-specific survival (12). Expression of PAK1 is also relevant to breast cancer survival, as its expression inhibits antiestrogenic action of tamoxifen in tamoxifen-sensitive breast cancer cell lines. Furthermore, PAK1 expression is linked to tamoxifen resistance and disease recurrence in primary breast carcinoma samples (7, 13).

The PAK1 gene product is a serine/threonine kinase involved in regulating cell morphology and motility through mediation of actin organization (14). In vitro expression of PAK1 is implicated in a broad array of signaling processes that correspond to breast cancer-related phenotypes, including cell migration, invasion, and anchorage-independent growth (15, 16). Regulation of anchorage-independent growth signaling and development of invasive breast carcinomas is mediated by proteins that directly interact with PAK1, including cyclin-D1 (CCND1), mitogen-activated protein kinase-1 (MAPK1), and RAS-homolog-family-member-A (RHOA) (9, 17). Overexpression of PAK1 has been shown to lead to focal adhesion complex disassembly, formation of lamellipodia and filopodia, and colocalization with actin filaments at the leading periphery of cells (14, 16, 18).

The Mammary carcinoma susceptibility (Mcs3) QTL, a 27.8 Mb segment on rat chromosome 1 that controls susceptibility to developing mammary carcinomas, contains sequence orthologous to human 11q13/14 (19). These orthologous sequences contain rat/human Pak1/PAK1 orthologs. Integrin-linked kinase (ILK), another Mcs3-nominated candidate gene (19), is on a different human chromosome than PAK1; however, a physical interaction between ILK and PAK1 has been documented (20). Similar to PAK1, expression of ILK is associated with breast cancer and 5-year survival (21). Both PAK1 and ILK expression are associated with female breast cancer (9, 15, 21), and rat orthologs of these genes are located at Mcs3. We sought to determine if expression of either rat Pak1 or Ilk is associated with rat Mcs3 genotype and DMBA-induced rat mammary carcinogenesis, using our integrated model of breast cancer susceptibility and development (22, 23).

METHODS

Congenic Strains and Animal Breeding

Wistar Furth.Copenhagen (WF.COP) congenic rat strains D and E used in this study are described in Le et. al. (19). Briefly, strain D contains a segment of Copenhagen (COP/NHsd) RNO1 from markers D1Rat277 (Chr1:134,120,332–134,120,480) to D1Rat321 (Chr1:171,716,519–171,716,714). Strain D is positive for the Mcs3 COP allele associated with decreased mammary cancer susceptibility. Strain E contains a segment of Copenhagen (COP/NHsd) RNO1 from markers D1Rat214 (Chr1:105,499,947-105,500,046) to D1Rat350 (Chr1:141,580,935-141,581,089). Strain E is negative for an independently acting Mcs3 decreased susceptibility allele. Rat chromosome physical positions are from UCSC rat genome assembly Rnor_6.0 (24, 25). Animals were housed in an Association for the Assessment and Accreditation of Laboratory Animal Care approved facility on a 12-h light/dark cycle. LabDiet 5001 Rodent Diet (PMI Nutrition International) and acidified water was provided ad libitum. All protocols involving animals were approved by the University of Louisville Institutional Animal Care and Use Committee.

DMBA Administration and Tissue Collection

At 50–55 days of age, 7,12-dimethylbenz(a)anthracene (DMBA Acros Organics, CAS No.: 57–97-6, 20 mg/mL in sesame oil) was given by single oral gavage (65 mg DMBA/kg body mass) to 10 WF.COP-RN01 strain D and 11 strain E females to induce mammary carcinogenesis. Age-matched females (9 of strain D and 12 of strain E) not exposed to DMBA were used as untreated controls. Four weeks following DMBA administration, the DMBA-exposed and age-matched nonexposed rats were euthanized by CO₂ asphyxiation. Abdominal-inguinal mammary glands were flash-frozen and stored at −80° before RNA extraction. Right thoracic mammary glands were formalin-fixed and paraffin-embedded (FFPE) for use in immunohistochemistry (IHC).

Quantitative PCR

Mammary tissue was homogenized in TRI-reagent (Molecular Research Center TR118) for total RNA extraction. Three molar sodium acetate (1/10 v/v) followed by a 100% ethanol (2.5× v/v) wash was used to remove potential solvent contaminants. Samples were then treated with Turbo DNAse (Life Technologies). Reverse transcription reactions, containing 1 μg RNA, 0.5× RNAsecure, 5 μM random hexamers (Thermo Fisher N8080127), 25 ng/μL oligo dT18 (Thermo Fisher 18418012), and 0.5 Mm 2′-deoxynucleoside 5′-triphosphate (dNTPs) (Thermo Fisher 10297018), were incubated for 5 min at 65°C. First-strand buffer (1×), 100 mM DTT, and 1 μL Superscript IV reverse transcriptase (Thermo Fisher 18090010) were added and incubated 5 min at 25°C, 1 h at 50°C, and 15 min at 70°C. Quantitative PCR reactions containing 1× TaqMan buffer A (Thermo Fisher), 3.5 mM MgCl2, 0.2 mM dNTP, 500 nM each forward and reverse primer, 200 nM target TaqMan gene-expression assays (Thermo Fisher) for Pak1 (Rn00664986_m1), Ilk (Rn00591471_m1), or Ribosomal protein lateral stalk subunit P2 (Rplp2) (Rn01479927_g1), 0.025 U/µl Taq Gold DNA pol (Thermo Fisher 18080240), and 50 ng cDNA were run at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. TaqMan gene-expression assay probe and primer sequences were: Pak1 FAM-ACTATCTGGACAGTTACCTTG with forward 5′-TGGTCATGAGGGAAAACAAAAA and reverse 5′-TCCATGACAACCCATAGCTCATC; Ilk FAM-CGGCTCAGGATTTT with forward 5′-GCAGGGACTTCAATGAGGAATG and reverse 5′-CGGAAGCACGTTAGGATGAGA; and Rplp2 VIC-CTGAATGGAAAGAATATTGAG with forward 5′-TGAACGACTCAACAAGGTCATCA and reverse 5′-CAACACCCTGAGCGATGACA. The manufacturer indicates that amplification efficiency is 100% ± 10% for TaqMan gene-expression assays (26). Amplification efficiencies for TaqMan assays ranged from 95% to 99% in our experiments. Quantitative PCR was conducted using an ABI Prism 7900HT Sequence Detection System with SDS v 2.3 software (Applied Biosystems).

Immunohistochemistry

Tissue was cut in 4-μm sections from FFPE blocks onto superfrost plus microscope slides (VWR). Slides were deparafinized in xylene and ethanol, boiled in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween, pH 6.0) for 20 min, washed in 1× TBS with 0.025% Triton X-100 for 10 min, and then blocked in 10% normal goat serum with 1% BSA in TBS for 2 h. Primary antibodies were diluted 1:100 in TBS with 1% BSA and kept with samples overnight at 4°C (anti-PAK1 Abcam131522, rabbit IgG Abcam 37415). Slides were visualized using an horseradish peroxidase/diaminobenzidine (HRP/DAB) detection IHC kit (Abcam ab64261), counterstained with hematoxylin, destained using xylene and ethanol, and coverslipped with ProLong Gold antifade mountant (Thermo Fisher P36930). Slides were imaged at ×40 magnification on an Aperio Imagescope 12.4 Software (Leica Biosystems).

Deidentified human breast disease clinical samples for IHC were from female patients seen at the University of Louisville (UofL) School of Medicine’s James Graham Brown Cancer Center (JG-BCC). Samples were obtained under an approved UofL IRB protocol to protect the privacy of research subjects.

The intensity of DAB staining of IHC samples was quantified using open-source ImageJ Fiji software (27). Corresponding tissue sections were quantified for Pak1 and control IgG. Microscopic images were submitted to the ImageJ plug-in vector H DAB, a function that color deconvolutes and outputs separate images of DAB staining and hematoxylin counterstaining. Mammary ductal structures were outlined in the deconvoluted DAB image output and mean intensity measured. The intensity value range is from 0, representing the darkest shade, to 255, representing a pure white, 8-bit image. Intensity values of Pak1 and IgG images were converted to optical density (OD), using the equation:

$$\text{OD }=\text{ log }\left(\text{max intensity}/\text{mean intensity}\right)$$

where maximum intensity is 255. Ratios of Pak1 OD to IgG OD for each image were used to determine relative Pak1 staining. Ratios were binned and assigned to a histogram profile using GraphPad Prism version 7.00. A Gaussian curve was fit to this plot. Samples with ratios that were >1 SD from the mean were counted as positive for Pak1 staining.

Sanger Sequencing and Strain Comparison

A reference rat Pak1 gene sequence was obtained from Ensembl ID ENSRNOG00000029784. Primer3 plus software was used to design PCR primers flanking Pak1 intron/exon splice sites and UTRs based on rat reference sequence RGSC 6.0/rn6. Primers and sequences are located in Supplemental Table S1, which is available at Mendeley Data (see http://dx.doi.org/10.17632/s5ywd3525d.1) (28). The UCSC genome browser in silico PCR tool was used to exclude off-target primers. Genomic DNA was extracted from rat spleen tissue, and PCR amplified at cycling conditions of 94°C for 2 min, 18 cycles of 94°C for 15 s and 68°C for 2 m, 22 cycles of 94°C for 15 s and 59°C for 2 m, and 59°C for 10 min. PCR samples were Sanger-sequenced using an ABI PRISM 3130xl Genetic Analyzer. Sequences were compared between WF, COP, and WF.COP-RN01 strain D rats. Identified variants were given dbSNP identifiers from NCBI and submitted to the European Variation Archive (EVA) project and Rat Genome Database ID. The database, UTRdb, contains curated 5′ and 3′UTRs of eukaryotic mRNAs (29) and was used to annotate functional untranslated region (UTR) elements. The miRNA database miRBase v22 was used to annotate miRNA binding sites within UTRs (30). When comparing sequences at intron/exon junctions, the consensus 5′ splice donor sequence GG(cut)GTRAGT and 3′ splice acceptor site CAG(cut)S were used, where R denotes any purine and S represents G or C. Branch sites were searched between 18 and 40 nucleotides upstream from the 3′ end of an intron using the consensus sequence YYRAY, where Y indicates a pyrimidine and R denotes any purine.

Data Handling and Statistical Analysis

Relative quantification, based on a standard curve, was used for qPCR. Mean quantities of triplicate qPCRs for each unknown sample were analyzed using two-way ANOVA, with log2 (Target quantity/Rplp2 quantity) as the dependent variable. Independent variables for comparing mammary gland transcript levels were Mcs3 genotype and DMBA exposure. A two-tailed, unpaired t test was performed post hoc. Pearson’s correlation coefficients were calculated for Pak1 and Ilk transcript levels from Mcs3 genotypes. The independent variables were Mcs3 genotype and DMBA exposure. All statistical analysis was done using GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla, California).

RESULTS

Quantitative PCR (qPCR) was used to compare Ilk and Pak1 mammary gland transcript levels between Mcs3 resistance-associated WF.COP congenic strain D and mammary carcinoma susceptible WF.COP strain E females following DMBAexposure. A time point of 4 wk post DMBA administration was used. This time represents a stage of early tumorigenesis before frank carcinomas are detectable in a DMBA-induced mammary carcinogenesis model. No effect of Mcs3 haplotype on expression of Ilk or Pak1 was observed at this time point, which was 12 wk of age (P > 0.05). Expression of Pak1 was significantly higher in rat mammary glands exposed to DMBA compared with unexposed age-matched controls (P = 0.02, Fig. 1A). There was no effect of either Mcs3 haplotype or DMBA exposure on mammary gland Ilk transcript levels (Fig. 1B). There was a correlation between Ilk and Pak1 mammary gland transcript levels in the mammary cancer-resistant WF.COP strain D unexposed control rats (r = 0.9349, P = 0.0007, Fig. 2C), but this correlation was lost with DMBA exposure (r = 0.5275, P = 0.1171, Fig. 2D). There was no correlation between Pak1 and Ilk in the mammary cancer-susceptible strain E with and without DMBA exposure.

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.

To determine if genetic variants in Pak1 protein-coding sequence existed between susceptible and Mcs3-resistance associated strains, we sequenced WF/NHsd, COP/NHsd, and Mcs3- resistance-associated WF.COP strain D. This revealed single nucleotide variants (SNVs) in exons 1, 2, 11, and 15 that differed between WF and COP or WF.COP strain D, but did not result in amino acid changes (Table 1). One variant was found in the 3′ splice site junction of exon 7; however, based on consensus splice sequence, this variant was not predicted to disrupt the splice site. We used the UTRdb database to predict three different upstream open reading frames (uORFs) in the 5′UTR and a polyadenylation signal (PAS) within the 3′UTR of Pak1. Variants located in Pak1 UTRs were not located in these elements. The database miRBase v22 was used to annotate miRNA-binding sites within rat Pak1 UTRs. No sites were identified in the 5′UTR. The 3′UTR contains an rno-miR-217-5p site; however, there were no variants between WF and COP or WF.COP strain D at this site.

Table 1.

Rat Pak1 sequence variants between strains

Marker Type	Type (Structure)	Structure Position(RGSC 6.0/rn6)	Variant Position RNO1	Variant	Referencea	WF/Hsd	COP/Hsd	WF.Cop Strain D	Effect on Information Metabolism	Predicted Biological Impact	SNV ID
SNV	Noncoding (5' UTR)	chr1:162817577–162817795	162817795	G/T	G	T	G	G	None expected	None identified	ss5327331130b14694838c
SNV	Coding (Exon 2)	chr1:162830944–162831133	162830964	C/T	C	T	C	C	Synonymous asp>asp	None identified	ss5327331131b14694839c
SNV	Splice site	chr1:162860673–162862490	162860683	T/C	T	C	C	C	Synonymous with splice site consensus	None identified	rs1065645844
SNV	Coding (Exon 11)	chr1:162871744–162871861	162871856	C/A	C	C	A	A	Synonymous arg>arg	None identified	rs106895892d
SNV	Noncoding (3' UTR)	chr1:162882087–162882685	162882562	C/T	C	C	T	T	None expected	None identified	rs198945644d

^aRattus norvegicus strain BN/SsNHsdMCW, ^bEuropean variation archive (EVA) project accession ID, ^cRat genome database ID, ^ddbSNP ID.

To determine if the DMBA-induced mammary carcinogenesis model is also a model of PAK1 expression during breast cancer development, IHC was performed on noncancerous rat mammary tissue exposed to DMBA. Figure 3 contains images of a rat mammary gland that displays positive Pak1 staining compared with an IgG background control. We quantitated relative Pak1 staining in mammary glands again at 4 wk post DMBA administration, as this represents an early stage of carcinogenesis. Overall, 4 out of 17 (24%) DMBA-exposed mammary glands were positive for Pak1 staining, whereas zero mammary glands out of seven (0%) taken from seven age-matched controls, not exposed to DMBA, stained positive for Pak1 (Table 2). Mammary gland tissue, positive for Pak1 staining at 4 wk post DMBA exposure, appeared histologically normal (Fig. 3).

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.

Table 2.

Rat mammary glands positive for Pak1 staining

Treatment	Total Samples	Pak1-Positive	Percent Positive
Unexposed controls	7	0	0
DMBA	17	4	24

DMBA, 7,12-dimethylbenz(a)anthracene; Pak1, p21-activated kinases.

To compare our findings in rat mammary glands with reported results of PAK1 positivity in human breast disease, IHC was used to visualize PAK1 expression in ER+/HER2− ductal carcinoma in situ (DCIS) and triple-negative (TN), ER+/HER2− and HER2+ invasive ductal breast carcinomas (IDC). Images of positive PAK1 staining, relative to IgG control, in TN, ER+/HER2−, HER2+ IDC, and ER+/HER2− DCIS are shown in Fig. 4. This was performed as a comparative experiment, with anticipation that our results would recapitulate those reported for PAK1 positivity in human breast disease. One out of six samples stained positive for PAK1 in both TN and HER2+ IDC cases. One out of four ER+/HER2− IDC samples displayed positive PAK1 staining, and one out of two ER+/HER2− DCIS samples were positive for PAK1. Overall, four breast disease tissue samples out of 18 (22%) analyzed were positive for PAK1 staining (Table 3).

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.

Table 3.

Female breast disease samples positive for PAK1 staining

Breast Disease Subtype	Total Samples	PAK1-Positive	Percent Positive
IDC TN	6	1	17
IDC HER2+	6	1	17
IDC ER+/HER2−	4	1	25
DCIS ER+/HER2−	2	1	50

Only a limited number of DCIS samples were available for study.

DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; TN, triple-negative.

DISCUSSION

We report that a DMBA-induced rat mammary carcinogenesis model of human breast cancer has utility to investigate a potential role for PAK1 at early stages of breast cancer development. Human PAK1 is located within the breast cancer-associated 11q13/14 amplicon. Human chromosome 11q13/14 amplification is reported to occur in 19.6% of breast tumors and is associated with poor prognosis (1–5, 32). Functional significance of PAK1 expression, due to 11q13/14 amplification, has been investigated using breast cancer cell lines in tissue culture and xenograft tumors (13, 14, 33–36). These in vitro studies provide insight into PAK1-driven progression of breast cancer cell lines, but do not indicate if aberrant PAK1 expression possesses a functional role during early disease pathogenesis, as normal tissue transitions toward a carcinoma. We did not uncover evidence supporting a hypothesis that Pak1 is an Mcs3 causal gene in this study. There are multiple Mcs3-nominated genes that will need to be studied in this context (19). We did discover an independent effect of DMBA exposure on Pak1 expression in mammary glands. Positive Pak1 expression was detected before pathological signs of disease developed in DMBA-exposed mammary glands. This suggests a potential early functional role of Pak1 in at least a subset of mammary glands suspected to be undergoing carcinogenesis.

We did not measure Pak1 protein and Pak1 transcript levels in the same samples; therefore, we have determined neither this relationship nor the mechanism for Pak1 positivity in a subset of rat mammary glands exposed to DMBA. We suspect that the increase in Pak1 transcript levels observed in mammary gland tissue following chemical exposure is due to either repopulation of the mammary gland with a high proportion of Pak1 expressing cells or increased transcription and/or amplification of the Pak1 locus in preexisting cells. Increased mRNA and protein stability are additional mechanisms that will need to be considered to determine the mechanism of Pak1 positivity.

Frequencies of PAK1-positive female breast disease samples found in samples we tested were consistent with a published study of PAK1 staining (9). We also detected positive PAK1 staining in a human DCIS sample. This matches previous findings of PAK1 positivity in human DCIS lesions using immunohistochemistry (37), and together suggest PAK1 expression may be involved in the progression of some female invasive breast carcinomas. This is concordant with a transcript profiling study of breast cancer progression that reported higher PAK1 transcript levels in human DCIS than in nondiseased breast epithelium (31). A study of breast cancer cell lines serially passed through xenografts in mice also reported a positive correlation between PAK1 expression and stage of premalignant progression (38). Our finding of elevated Pak1 expression in chemical carcinogen-induced nondiseased rat mammary glands further suggests that PAK1 has a role during early tumorigenesis of female breast cancer, potentially before the development of premalignant lesions.

Rodent models have long been used to explore gene function during cancer progression (39). The only published rodent study of PAK1 and breast disease, to the best of our knowledge, used transgenic mice expressing an activated form of Pak1 (40). These authors demonstrated a role for Pak1 in mammary lesion and tumor formation; however, the expression of an activated-Pak1 transgene was dependent upon a pituitary gland isograft. Few mammary tumors (MTs) developed in this model over a long latency period of 2 yr. We propose that laboratory rats, well-known to serve as a female breast cancer-relevant in vivo system, could be used to study Pak1-mediated mechanisms using a shorter time interval. Palpable mammary tumors typically develop in susceptible rat strains around 6 wk following a single DMBA exposure. A laboratory rat model recapitulates a natural course of breast cancer development, which provides an in vivo model to study Pak1-mediated carcinogenesis. The laboratory rat is an excellent model of human breast cancer, as induced rat mammary carcinomas display the same histopathological stages and features of malignant breast carcinomas, including epithelial ductal cell origin, progression, and an array of hormone responsive and nonresponsiveness (23, 41–46). The mammary carcinoma-susceptible rat strains used in this study develop on average seven mammary tumors per rat upon DMBA induction, which is ∼0.58 tumors per mammary gland (MG). Our finding that 24% of DMBA-induced mammary glands were Pak1-positive is extrapolated to estimate Pak1 positivity in 14% of rat mammary tumors (MTs) (0.58 MT/MG × 0.24 Pak⁺-MGs). This is in line with the 15%–25% PAK1 positivity of human breast carcinoma samples in this study, and experimentally demonstrates similar Pak1/PAK1 expression patterns in mammary/breast disease. Therefore, a rat model allows for experimentation and pathway analyses of Pak1 in the context of natural disease initiation, development, and progression that would complement human breast cancer studies.

In this study, we discovered that DMBA-induction of mammary carcinogenesis increased Pak1 transcript levels in mammary gland tissue that appeared to be morphologically normal. We also reported that Pak1 protein expression in rat mammary glands mirrored what we and others have found in human breast DCIS and IDC samples. These findings of rat/human Pak1/PAK1 staining at early stages of rat mammary carcinogenesis and premalignant stages of female breast cancer warrant further functional and diagnostic studies of PAK1 expression during early stages of rat mammary and human breast cancer development.

GRANTS

This work is supported by The University of Louisville Integrated Programs in Biomedical Sciences and the Department of Biochemistry and Molecular Genetics (stipend support to E.L. Duderstadt). The University of Louisville Cancer Education Program (R25 CA134283) partially supported this work.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.L.D. and D.J.S. conceived and designed research; E.L.D., S.A.M., and D.J.S. performed experiments; E.L.D., S.A.M., M.A.S., and D.J.S. analyzed data; E.L.D., M.A.S., and D.J.S. interpreted results of experiments; E.L.D. prepared figures; E.L.D. and D.J.S. drafted manuscript; E.L.D. and D.J.S. edited and revised manuscript; E.L.D., S.A.M., M.A.S., and D.J.S. approved final version of manuscript.