Breast Cancer Res Treat (2012) 133:909–916 DOI 10.1007/s10549-011-1868-5

PRECLINICAL STUDY

Expression of SPRR3 is associated with tumor cell proliferation in less advanced stages of breast cancer Jin Cheon Kim • Jong Han Yu • Yoon Kyung Cho • Choon Sik Jung • Sei Hyun Ahn • Gyungyub Gong • Yong Sung Kim • Dong-Hyung Cho

Received: 12 August 2011 / Accepted: 31 October 2011 / Published online: 11 November 2011 Ó Springer Science+Business Media, LLC. 2011

Abstract Small proline rich repeat protein 3 (SPRR3), a member of the SPRR family of cornified envelope precursor proteins, is a marker for terminal squamous cell differentiation. Previously, this laboratory showed that SPRR3 is strongly upregulated in colorectal tumors, and is involved in the tumorigenesis. The current study was performed to investigate the expression status and effect of SPRR3 in breast cancers (BCs). SPRR3 expression was examined by immunohistochemistry in 241 tumor samples from BC patients. SPRR3 was overexpressed in more than half of all BC samples. SPRR3 overexpression was significantly associated with less advanced stage (0–1 vs. II–III) and the absence of lymph node metastasis (P = 0.004 and 0.013, respectively). HER2/neu overexpression was closely J. C. Kim (&)  J. H. Yu  Y. K. Cho  C. S. Jung  S. H. Ahn Department of Surgery, University of Ulsan College of Medicine, Seoul, Republic of Korea e-mail: [email protected] J. C. Kim  J. H. Yu  Y. K. Cho  C. S. Jung  S. H. Ahn  Y. S. Kim  D.-H. Cho Institute of Innovative Cancer Research, Asan Medical Center, 86 Asanbyeongwon-gil, Sonpa-gu, Seoul 138-736, Republic of Korea G. Gong Department of Pathology, University of Ulsan College of Medicine, Seoul, Republic of Korea Y. S. Kim Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong Yuseong-ku, Daejeon 305-333, Republic of Korea D.-H. Cho (&) Graduate School of East–West Medical Science, Kyung Hee University, Gyeonggi-Do, Republic of Korea e-mail: [email protected]

correlated with SPRR3 overexpression in a multivariate analysis (OR, 3.23, P = 0.017). To assess the influence of SPRR3 on cell proliferation and related signaling pathways, SPRR3-transfected clones from the SPRR3-negative T-47D human BC cell line were generated. Among the total of six SPRR3-overexpressing clones, five showed marked proliferation compared with SPRR3-nonexpressing control cells from day 3 of culture (P \ 0.001). The SPRR3-overexpressing BC clones showed increased phosphorylation of AKT and MDM2, p21 overexpression, and p53 downregulation. Furthermore, phosphorylation of MEK and MAPK was markedly increased. This study demonstrates that SPRR3 promotes BC cell proliferation by enhancing p53 degradation via the AKT and MAPK pathways and is, therefore, a potential novel therapeutic target for less advanced stages of BC. Keywords

Breast cancer  SPRR3  Tumorigenesis  p53

Abbreviations AKT Serine/threonine protein kinase BC Breast cancer HR CI Confidence interval CRC Colorectal cancer DFS Disease-free survival ER Estrogen receptor ESCC Esophageal squamous cell cancer HER2/neu Human epidermal growth factor receptor 2 HR Hazard ratio MAPK Mitogen-activated protein kinase MDM2 Murine double minute 2 MEK MAPK/extracellular signal-regulated kinase OR Odds ratio OS Overall survival PI3K Phosphatidylinositol 30 kinase

123

910

PR RAF REMARK siRNA SPRR3 TNBC

Breast Cancer Res Treat (2012) 133:909–916

Progesterone receptor Rapidly accelerated fibrosarcoma Reporting recommendations for tumor marker prognostic studies Small interfering ribonucleic acid Small proline rich repeat protein 3 Triple negative BC

Introduction Breast cancer is the most common cancer diagnosed among women worldwide and is the second most common cancer among Korean women [1, 2]. Early detection and improved postoperative adjuvant therapy including chemoradiotherapy and anti-hormone therapy have enhanced survival outcome. In addition to these modalities, target molecules involved in the tumorigenesis and progression have been investigated to develop efficient anti-cancer drugs, specifically referring to their gene mutations. For example, survival is dramatically improved with appropriate chemotherapy combined with the monoclonal antibody trastuzumab which specifically targets HER2/neu [3]. Several randomized trials using different chemotherapy regimens combined with trastuzumab showed significantly improved survival, reducing risk of recurrence by 33–46% [3–5]. The estrogen receptor and HER2/neu signaling pathways are the major influences on cell proliferation and survival in approximately 85% BCs [6]. The gene encoding HER2/neu, one of a family of four closely related membrane-bound receptor tyrosine kinases, is amplified in some human BC cell lines, and this amplification provides potent proliferation and anti-apoptosis signals. Activation of Her2/neu via ligand binding and intracellular crosstalk, however, can only occur when it forms functional dimers with other family members [7]. Major pathways involved in signal transduction, including RAF/MAPK and PI3K/ AKT, ultimately affect cell proliferation and survival. The PI3K/AKT pathway is activated in 70% of BCs and is associated with aggressive features such as high histological grade, and basal-like and HER2/neu phenotypes, resulting in poor clinical outcome [8, 9]. Growth factors, such as epidermal growth factor and insulin-like growth factor, activate AKT, which, in turn, phosphorylates and activates MDM2 leading to downregulation of p53 and cell survival (the survival pathway) [10]. Small proline rich repeat protein 3 (SPRR3), a member of the SPRR family of cornified envelope precursor proteins, is a marker for terminal squamous cell differentiation, but also functions in non-squamous tissues [11]. SPRR3 expression in certain lesions such as benign papilloma, Bowen’s disease and squamous cell carcinoma

123

suggests that SPRR3 is associated with malignant conversion or progression during epidermal carcinogenesis [12]. A correlation between SPRR3 and a specific molecular pathway connecting Wnt and RAS pathways has been demonstrated from gene expression profiles of sporadic colorectal cancers (CRCs) [13]. Extension of this study demonstrated that SPRR3 overexpression accelerates CRC cell proliferation and p53 downregulation, and may be associated with colorectal tumorigenesis [14]. In the present study, SPRR3 was shown to promote BC cell proliferation and its overexpression was significantly associated with less advanced stages of disease. Overexpression of SPRR3 in a BC cell line led to an increase in AKT and MDM2 phosphorylation, p21 overexpression and p53 downregulation. MEK and MAPK phosphorylation were also increased in SPRR3-overexpressing clones.

Materials and methods Patient enrollment, eligibility and treatment For the initial screening of SPRR3 expression, 19 BC tissue samples were randomly chosen from the Asan Medical Center (Seoul, Korea) archive. For the clinical validation, 241 patients with available tumor samples were retrospectively recruited from a total of 292 consecutive BC patients including the same number of patients (73 patients) in the respective stage of 0–III (Table 1). The eligibility criteria were as follows: curatively resected (R0) infiltrating ductal or lobular carcinoma of the breast; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; age of 70 years or less. Patients with a previous history of any cancer or familial BC were excluded. The extent of the disease was assessed by clinical examination; excisional or core needle biopsy; estrogen and progesterone receptor (ER and PR) and Her2/neu assays; chest radiography and mammography. Among the 241 patients, 152 received adjuvant chemotherapy consisting of adriamycin, cyclophosphamide and taxane, of whom 122 also received adjuvant radiotherapy. Tamoxifen or aromatase inhibitor anti-hormone therapy was used in 115 of the 241 patients. Patient follow-up Patients underwent a post-operative follow-up assessment every 6 months for the first 5 years and then annually thereafter for a further 5 years; the mean follow-up period was 60 (range, 5–70) months. Evaluations included clinical examination, routine blood chemistry, chest radiography, and CT of the chest and abdomen or radionuclide bone scan. Locoregional recurrence was defined as tumor regrowth in

Breast Cancer Res Treat (2012) 133:909–916 Table 1 Patients’ tumor characteristics

ER estrogen receptor, PR progesterone receptor a

Cancer staging according to the American Joint Committee on Cancer (7th Ed., 2010)

b

Tumors showing ER-, PRand Her2/neu-

911

Parameters

No. of patients (%), n = 241

Stagea, 0/I/II/III

48/66/71/56 (19.9/27.4/29.5/23.2)

T-category, Tis/1/2/3/4

49/93/86/8/5 (20.3/38.6/35.7/3.3/2.1)

Missing no.

N-category, 0/1/2/3

136/52/30/23 (56.4/21.6/12.4/9.5)

Grade, 1/2/3

14/94/115 (6.3/42.2/51.6)

18

Lymphovascular invasion

77 (37)

33

?

ER

122 (50.6)

PR?

123 (51)

Her2/neu?

90 (37.7)

2

Triple-b

57 (23.8)

2

Recurrence, locoregional/distant

11/29 (4.6/12)

the ipsilateral breast (local recurrence) or regional lymph nodes (regional recurrence), whereas distant recurrence was defined as any other recurrence based on clinical and/or radiographic findings. The primary endpoints were recurrence, disease-free survival (DFS) and overall survival (OS). All patients provided written informed consent. The study protocol was approved by the Institutional Review Board for Human Genetic and Genomic Research (registration no.: 2009-0091) in accordance with the Declaration of Helsinki. Cell line, transfection and proliferation assay The T-47D ductal BC cell line, obtained from the American Type Culture Collection (Manassas, VA), was cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 incubator. SPRR3 cDNA PCR-amplified and subcloned into hemagglutinin (HA)-tagged pcDNA3 (pHA-SPRR3) (Invitrogen) was used to generate stable transfectants, as previously described [14]. T-47D cells were transfected with pHA-SPRR3 using lipofectamine 2000 (Invitrogen). Stably expressing cells, confirmed by Western blot analysis, were selected with G418 (1 mg/ml) for 10 days and then cloned (T-47D/SPRR3). A control T-47D clone was established using an empty expression vector, pcDNA3.1 (Invitrogen). The SPRR3 small interfering RNA (siRNA) (50 -CAGACAAGCCCUUGAGAA30 ) and non-targeting scrambled siRNA (50 -CCUACGCCACCAAUUUCGU-30 ) were synthesized by Bioneer (Daejeon, Republic of Korea) and transfected into T-47D cells. Knock-down efficiency was assessed by Western blot analysis with anti-SPRR3 antibody. T-47D cells were seeded onto a 96-well plate and the cell proliferation rate was measured by a plate reader (absorbance at 450 nm) after incubation with the CellCounting Kit 8 (CCK8) reagent (10 ll) (Dojindo Laboratories, Kumamoto, Japan) for 2 h. T-47D clones were cultured until the day 4 and examined every day using triplicates of each clone.

Western blot and immunohistochemistry Protein from the T-47D clones was extracted in 29 Laemmli sample buffer (BioRad, Hercules, CA), followed by SDS–PAGE and transfer to polyvinylidene difluoride membranes. After blocking with skimmed milk in Trisbuffered saline Tween-20, membranes were incubated with the following specific primary antibodies: anti-SPRR3 antibody from Proteintech (Chicago, IL); anti-phosphoAKT, anti-phospho-MDM, anti-MEK, anti-phospho-MEK, anti-MAPK (ERK1/2), anti-phospho-MAPK (ERK1/2), and anti-p53 antibodies from Cell Signaling Technology (Beverly, MA); anti-p21 from BD Pharmingen (San Jose, CA); anti-Actin antibody from Chemicon International (Temecula, CA) and anti-HA antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). All Western blot assays were performed in quadruplicate. For the construction of tissue microarrays paraffinembedded tissue cores were arrayed on recipient blocks using precision microarray equipment (Beecher Instruments, Sun Prairie, WI). Each sample was arrayed in triplicate to minimize tissue loss and to reduce skewed tissue sampling as a result of tumor heterogeneity. Tissue sections and arrays were checked for representative morphology after staining with hematoxylin and eosin. Tissue array blocks were subjected to the labeled streptavidin– biotin immunohistochemical staining method with a DAKO LSABÒ kit (DAKO, Carpinteria, CA), and monoclonal antibodies against SPRR3 (Proteintech), ER, PR (Diona, Seoul, Korea), and HER2/neu (DAKO). Cytoplasmic (SPRR3) or nuclear (ER and PR) expression was classified into two categories; negative expression at B10% and positive expression at [10% staining cells. HER2/neu immunostaining was considered positive when overexpression of membranous staining (classified as 3?) was observed in at least 10% of tumor cells, whereas cases classified as having 0–2? membranous staining were

123

912

regarded as negative. Stained tissue sections and arrays were analyzed by two pathologists. Statistical methods and accomplishment of the REMARK guidelines Under the preliminary result of a difference of SPRR3 overexpression rate between normal and tumor tissues of 40%, the Altman’s nomogram was used to determine the sample size (*240 patients) of the present study and insure 85% power to detect clinical outcome. The clinicopathological variables between patients with positive and negative protein expression were compared using the two-sided Fisher’s exact test. Potential variables were verified by multivariate analysis using binary logistic regression. OS and DFS were compared using the Kaplan–Meier method with the log-rank test. Statistical significance was defined as a P-value \0.05. All calculations were performed using SPSS software (ver. 19, SPSS Inc., Chicago, IL). The current study observed the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelines including study design, planned hypotheses, sample acquisition, assay methods and interpretation, and statistical analysis, as much as possible [15].

Results Recurrence and survival outcome Locoregional and distant recurrences were significantly associated with advanced stage, T- or N-category, and high combined histologic grade (P B 0.003) (Table 2). Among these parameters, advanced T- (T3–4) and N-categories (N1–3) were significantly correlated with recurrences in a multivariate analysis (OR, 7 and 6.42; P = 0.013 and 0.035, respectively). Accordingly, OS and DFS were significantly reduced with these recurrence-related parameters (P B 0.004). Patients with advanced T-categories (T3–4) had a more than 4-fold reduction in OS and DFS compared to those with Tis-T2 in a Cox regression analysis (HR, 4.65 and 4.53; 95% CI, 1.92–11.25 and 1.93–10.65; P = 0.001, respectively). In addition, DFS was significantly reduced in patients with lymph node metastasis compared to those without (HR, 3.18; 95% CI, 1.09–9.24; P = 0.034). Correlation of SPRR3 expression with clinicopathologic parameters Among the 19 breast cancer patients who were screened initially, 10 (53%) had strong expression of SPRR3 in the tumor compared with adjacent normal breast tissue (Fig. 1a). Similarly in the validation cohort of 241 patients,

123

Breast Cancer Res Treat (2012) 133:909–916

SPRR3 was overexpressed in the tumor samples of 133 patients (55.2%) (Fig. 1b), while it was downregulated in the remainder (Fig. 1c). SPRR3 overexpression was significantly associated with less advanced stage (0–1 vs. II– III), HER2/neu overexpression, absence of lymph node metastasis, and lack of triple negative (ER-, PR- and Her2/neu-) BCs (TNBC)(P B 0.019) (Table 3). HER2/neu overexpression was also closely correlated with SPRR3 overexpression in a multivariate analysis using potential parameters, i.e., stage, N-category, ER and PR status, HER2/neu expression, and triple- status (OR, 3.23; 95% CI, 1.24–8.44; P = 0.017). The status of SPRR3 expression did not associate with recurrence rate (overexpression vs. downregulation: 14.3 vs. 19.4%, P = 0.301), 5-year OS (overexpression vs. downregulation: 87.2 vs. 87%, P = 0.863), and 5-year DFS (overexpression vs. downregulation: 82.7 vs. 78.5%, P = 0.566). Effect of SPRR3 overexpression on BC cell proliferation As SPRR3 was overexpressed in more than half of BC samples, the effect of SPRR3 on the proliferation of BC cells was investigated. SPRR3-overexpressing clones were established from the SPRR3 negative, HER2/neu positive T-47D human BC cell line. Six SPRR3-overexpressing clones were established, and compared with SPRR3 nonexpressing T-47D cells, five of the clones proliferated markedly from day 3 of culture (P \ 0.001) (Fig. 2). Effect of SPRR3-overexpression on AKT and MAPK signaling pathways and p53 To determine the functional implication of SPRR3 in BC cell proliferation, the possible involvement of AKT and MAPK pathways in SPRR3-overexpressing cells was investigated (Fig. 3a). AKT and MAPK both play pivotal roles in fundamental cellular functions such as cell proliferation, migration, and apoptosis. The six SPRR3-overexpressing clones displayed increased AKT phosphorylation and subsequently expressed increased but variable amounts of phosphorylated MDM2, while concurrently overexpressing p21. Compared to T-47D control cells, p53 expression was completely downregulated in SPRR3-overexpressing T-47D cells, while MEK and MAPK phosphorylation was markedly increased. To investigate the effect of downregulated SPRR3 in BC cells, SPRR3-expressing T-47D clones were transiently transfected with either non-targeting scrambled siRNA or SPRR3-specific siRNA (Fig. 3b). Phosphorylation of MEK and MAPK was markedly reduced in the siSPRR3-treated cells, whereas p53 expression was restored to some extent. Although AKT phosphorylation and p21 expression were

Breast Cancer Res Treat (2012) 133:909–916

913

Table 2 Association between recurrence and breast cancer parameters Parameters

No. of tumors with recurrence (%)

P-valuea

OR

95% CI

Stagec, 0 ? I vs. II ? III

4/114 (3.5) vs. 36/127 (28.3)

<0.001

2.22

0.25–19.6

0.472

T-category, Tis-2 vs. T3–4

31/228 (13.6) vs. 9/13 (69.2)

<0.001

7

1.51–32.52

0.013

N-category, N0 vs. N1-3

6/136 (4.4) vs. 34/105 (32.4)

<0.001

6.42

1.14–36.31

0.035

d

P-valueb

Histologic grade , G1–2 vs. G3

10/108 (9.3) vs. 26/115 (22.6)

0.01

1.55

0.62–3.90

0.351

Lymphovascular invasion, no vs. yes

15/131 (11.5) vs. 22/77 (28.6)

0.003

0.91

0.37–2.24

0.838

ER- vs. ER?

24/119 (20.2) vs. 16/122 (13.1)

0.167

PR- vs. PR?

24/118 (20.3) vs. 16/123 (13)

0.166

HER2/neu- vs. HER2/neu?

23/149 (15.4) vs. 17/90 (18.9)

0.481

Triple-e vs. lack of triple-

11/57 (19.3) vs. 29/182 (15.9)

0.542

ER estrogen receptor, PR progesterone receptor, OR odds ratio, CI confidence interval. Bold font, P \ 0.05 a

All parameters were compared by the two-sided Fisher’s exact test

b

Multivariate analysis by binary logistic regression using potential parameters, i.e., stage, T- and N-categories, histologic grade, and lymphovascular invasion

c

Cancer staging according to the American Joint Committee on Cancer (7th ed., 2010)

d

Nottingham combined histologic grade

e

Tumors showing ER-, PR-, and Her2/neu-

Fig. 1 Expressions of SPRR3 in breast tissues (C cancer tissue, N corresponding normal tissue) by Western blots (a). Representative photomicrographs of immunohistochemical staining for SPRR3 in breast cancer tissues, showing overexpression, (b) and downregulation, (c). Hematoxylin was used for nuclear counterstaining. Bar 20 lm

not apparently suppressed in the siSPRR3-treated cells, their expression patterns were similar to those of the control scrambled siRNA-treated cells.

Discussion SPRR3 promoted BC cell proliferation and it was strongly upregulated in the tumors of more than half of the BC patients. The biological characteristics of SPRR3-expressing tumors appear to vary depending on histological type. One study reports that the ratio of expression of GATA-binding protein 6 (GATA6), which is overexpressed in colon cancer cells and other cancers, to SPRR3 can significantly

discriminate between normal esophageal epithelium, Barrett’s dysplasia and adenocarcinoma [16]. Loss of SPRR3 is suggested to be a harbinger of early esophageal malignant transformation, and observation of its downregulation may be valuable in diagnosis and assessment of response to chemoradiotherapy in esophageal squamous cell cancer (ESCC) [16–18]. In contrast to the pattern seen in ESCC, in CRC SPRR3 overexpression not only causes accelerated tumor cell proliferation but it is also associated with lymphovascular invasion [14]. Interestingly, in the current study SPRR3 overexpression was closely correlated with less advanced stages and the absence of lymph node metastasis in BC patients; however, there was no correlation with incidence of recurrence or survival duration. These paradoxical findings

123

914

Breast Cancer Res Treat (2012) 133:909–916

Table 3 Association between SPRR3 expression and breast cancer parameters Parameters

No. of tumors with SPRR3 overexpression (%)

P-valuea

OR

95% CI

P-valueb

Stagec, 0 ? I vs. II ? III

74/114 (64.9) vs. 59/127 (46.5)

0.004

0.49

0.19–1.28

0.146

T-category, Tis-2 vs. T3-4

128/228 (56.1) vs. 5/13 (38.5)

0.258

N-category, N0 vs. N1-3

85/136 (62.5) vs. 5/19 (45.7)

0.013

0.97

0.37–2.51

0.964

Histologic grade , G1–2 vs. G3

61/108 (56.5) vs. 61/115 (53)

0.687

Lymphovascular invasion, no vs. yes

71/131 (54.2) vs. 39/77 (50.6)

0.667

ER- vs. ER?

59/119 (49.6) vs. 74/122 (60.7)

0.093

0

PR- vs. PR?

58/118 (49.2) vs. 75/123 (61)

0.071

0

HER2/neu- vs. HER2/neu?

74/149 (49.7) vs. 57/90 (63.3)

0.045

3.23

1.24–8.44

0.017

Triple-d vs. lack of triple-

23/57 (40.4) vs. 108/182 (59.3)

0.015

0.54

0.16–1.83

0.539

d

1 1

ER estrogen receptor, PR progesterone receptor, OR odds ratio, CI confidence interval, Bold font, P \ 0.05 a

All parameters were compared by the two-sided Fisher’s exact test

b

Multivariate analysis by binary logistic regression using potential parameters, i.e., stage, N-category, ER and PR status, HER2/neu expression, and Triple- status

c

Cancer staging according to the American Joint Committee on Cancer (7th ed., 2010)

d

Tumors showing ER-, PR-, and Her2/neu-

Fig. 2 SPRR3 increases cell proliferation. The cell proliferation rates of control T-47D cells and T-47D cells stably expressing SPRR3 (T47D/SPRR3) were measured by a CCK8 cell proliferation assay. Among six SPRR3-overexpressing clones, five clones (#1–5) proliferated remarkably from the culture day 3 compared with control T-47D cells (P \ 0.001)

suggest that SPRR3 overexpression may associate with the expression of other molecules that determine aggressive behavior in BC. In the multivariate analysis, HER2/neu overexpression, identified in 38% of the patients, was approximately threetimes more frequent in tumors with SPRR3 overexpression. HER2/neu is a membrane-bound receptor tyrosine kinase proto-oncogene and its overexpression has been identified in 10–34% of invasive breast cancers [7]. HER2/neu overexpression has consistently been associated with high tumor grade, DNA aneuploidy, high cell proliferation rate, negative ER and PR, TP53 mutation, topoisomerase IIa

123

Fig. 3 The expression changes of several signaling molecules involved in the cell cycle or cell proliferation were evaluated by Western blot analysis in SPRR3-expressing T-47D cells. The AKT and MEK signaling pathway were activated in SPRR3-expressing cells a Phosphorylations of MEK and MAPK were reduced in siSPRR3-treated T-47D cells, b Stable SPRR3 expression was detected with anti-HA antibody and actin was used as an internal loading control

amplification and alterations in a variety of other molecules facilitating invasiveness and metastasis [7, 19]. Consequently, concomitant Her2/neu overexpression could explain the correlation found between SPRR3 overexpression and less advanced tumors but without survival gain. Furthermore, in the current study, SPRR3 downregulation was associated with TNBC. TNBC is another aggressive phenotype characterized by lack of or minimal expression of ER and PR as well as an absence of HER2/

Breast Cancer Res Treat (2012) 133:909–916

neu overexpression [20]. The more advanced tumors with SPRR3 downregulation in our patients could be those with the aggressive TNBC phenotype. TNBC, characterized by an aggressive clinical course and poor survival, is not amenable to hormone therapy or HER2/neu-targeted agents such as trastuzumab [20, 21]. All the SPRR3-overexpressing clones generated from the T-47D cell line had downregulated p53 expression, accompanied by AKT and MDM2 phosphorylation and p21 overexpression. AKT is known to regulate cell growth by modulating p21, p27, GSK3b, and MDM2 [22–24]. AKT promotes p53 degradation through the phosphorylation of MDM2, and p53 controls cell proliferation by inducing either growth arrest or apoptosis [24–26]. MDM2 binds to the N-terminal region of p53 and represses its activity either by promoting p53 exportation to the cytoplasm or by blocking p53 transcriptional activation [10]. In the current study, MEK and MAPK phosphorylation was markedly increased in SPRR3-overexpressing cells. Conversely, phosphorylation of MEK and MAPK was significantly reduced in siSPRR3-treated cells. Signaling through the RAF-MEK pathway, the first MAPK cascade to be identified, mediates developmental processes and tumorigenesis [27]. Growth factors and mitogens transmit signals via their receptors through this cascade to regulate gene expression and prevent apoptosis [28]. In the current study, SPRR3 has been shown to promote BC cell proliferation by enhancing p53 degradation via the AKT and MAPK pathways. SPRR3 is, therefore, a potential novel therapeutic target for less advanced BCs. The close correlation between SPRR3 and HER2/neu expression revealed by this study warrants further investigation to clarify the specific pathology of this subset of BCs. Acknowledgments This study was supported by grants to J.C.K. from the Asan Institute for Life Sciences (2011-069), the Korea Health 21 R&D Project (A062254) and the Center for Development and the Commercialization of Anti-Cancer Therapeutics (A102059), Ministry of Health, Welfare, and Family Affairs, Republic of Korea. Conflict of interest peting interests.

915

4.

5.

6. 7.

8.

9.

10. 11.

12.

13.

14.

The authors declare that they have no com15.

16.

References 1. Ko SS, Korean Breast Cancer Society (2005) Chronological changing patterns of clinical characteristics of Korean breast cancer patients during 10 years (1996–2006) using nationwide breast cancer registration on-line program: biannual update. J Surg Oncol 98:318–323 2. Parkin DM, Bray FI, Devesa SS (2001) Cancer burden in the year 2000. The global picture. Eur J Cancer 37:S4–S66 3. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, Alanko T, Kataja V, Asola R, Utriainen T, Kokko R, Hemminki A, Tarkkanen M,

17.

18.

Turpeenniemi-Hujanen T, Jyrkkio¨ S, Flander M, Helle L, Ingalsuo S, Johansson K, Ja¨a¨skela¨inen AS, Pajunen M, Rauhala M, Kaleva-Kerola J, Salminen T, Leinonen M, Elomaa I, Isola J, Investigators FinHer Study (2006) Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer. N Engl J Med 354:809–820 Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, La´ng I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ru¨schoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD, Adjuvant Herceptin, (HERA) Trial Study Team (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659–1672 Kaufman PA, Swain SM, Pisansky TM, Fehrenbacher L, Kutteh LA, Vogel VG, Visscher DW, Yothers G, Jenkins RB, Brown AM, Dakhil SR, Mamounas EP, Lingle WL, Klein PM, Ingle JN, Wolmark N (2005) Trastuzumab plus adjuvant chemotherapy for operable HER2positive breast cancer. N Engl J Med 353:1673–1684 Gutierrez C, Schiff R (2011) HER2: biology, detection, and clinical implications. Arch Pathol Lab Med 135:55–62 Ross JS, Slodkowska EA, Symmans WF, Pusztai L, Ravdin PM, Hortobagyi GN (2009) The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine. Oncologist 14:320–368 Lo´pez-Knowles E, O’Toole SA, McNeil CM, Millar EK, Qiu MR, Crea P, Daly RJ, Musgrove EA, Sutherland RL (2010) PI3K pathway activation in breast cancer is associated with the basallike phenotype and cancer-specific mortality. Int J Cancer 126:1121–1131 Castaneda CA, Cortes-Funes H, Gomez HL, Ciruelos EM (2010) The phosphatidyl inositol 3-kinase/AKT signaling pathway in breast cancer. Cancer Metastasis Rev 29:751–759 Lacroix M, Toillon RA, Leclercq G (2006) p53 and breast cancer, an update. Endocr Relat Cancer 13:293–325 Tesfaigzi J, Th’ng J, Hotchkiss JA, Harkema JR, Wright PS (1996) A small proline-rich protein, SPRR1, is upregulated early during tobacco smoke-induced squamous metaplasia in rat nasal epithelia. Am J Respir Cell Mol Biol 14:478–486 De Heller-Milev M, Huber M, Panizzon R, Hohl D (2000) Expression of small proline rich proteins in neoplastic and inflammatory skin diseases. Br J Dermatol 143:733–740 Kim JC, Kim SY, Roh SA, Cho DH, Kim DD, Kim JH, Kim YS (2008) Gene expression profiling: canonical molecular changes and clinicopathological features in sporadic colorectal cancers. World J Gastroenterol 14:6662–6672 Cho DH, Jo YK, Roh SA, Na YS, Kim TW, Jang SJ, Kim YS, Kim JC (2010) Upregulation of SPRR3 promotes colorectal tumorigenesis. Mol Med 16:271–277 McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM (2005) Reporting recommendations for tumor marker prognostic studies (REMARK). J Natl Cancer Inst 97:1180–1184 Kimchi ET, Posner MC, Park JO, Darga TE, Kocherginsky M, Karrison T, Hart J, Smith KD, Mezhir JJ, Weichselbaum RR, Khodarev NN (2005) Progression of Barrett’s metaplasia to adenocarcinoma is associated with the suppression of the transcriptional programs of epidermal differentiation. Cancer Res 65:3146–3154 Luthra R, Wu TT, Luthra MG, Izzo J, Lopez-Alvarez E, Zhang L, Bailey J, Lee JH, Bresalier R, Rashid A, Swisher SG, Ajani JA (2006) Gene expression profiling of localized esophageal carcinomas: association with pathologic response to preoperative chemoradiation. J Clin Oncol 24:259–267 Kimos MC, Wang S, Borkowski A, Yang GY, Yang CS, Perry K, Olaru A, Deacu E, Sterian A, Cottrell J, Papadimitriou J, Sisodia

123

916

19. 20.

21.

22.

23.

Breast Cancer Res Treat (2012) 133:909–916 L, Selaru FM, Mori Y, Xu Y, Yin J, Abraham JM, Meltzer SJ (2004) Esophagin and proliferating cell nuclear antigen (PCNA) are biomarkers of human esophageal neoplastic progression. Int J Cancer 111:415–417 Yarden Y (2001) Biology of HER2 and its importance in breast cancer. Oncology 61(suppl 2):1–13 Pal SK, Childs BH, Pegram M (2011) Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat 125: 627–636 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA (2007) Triplenegative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res 13:4429–4434 Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA (1995) Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785–789 Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC (2001) Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol 3:245–252

123

24. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC (2001) HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 3:973–982 25. Ogawara Y, Kishishita S, Obata T, Isazawa Y, Suzuki T, Tanaka K, Masuyama N, Gotoh Y (2002) Akt enhances Mdm2-mediated ubiquitination and degradation of p53. J Biol Chem 277: 21843–21850 26. Agrawal A, Yang J, Murphy RF, Agrawal DK (2006) Regulation of the p14ARF-Mdm2–p53 pathway: an overview in breast cancer. Exp Mol Pathol 81:115–122 27. Tartaglia M, Gelb BD (2010) Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms. Ann NY Acad Sci 1214:99–121 28. McCubrey JA, Steelman LS, Chappell WH, Abrams SL, Wong EW, Chang F, Lehmann B, Terrian DM, Milella M, Tafuri A, Stivala F, Libra M, Basecke J, Evangelisti C, Martelli AM, Franklin RA (2007) Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta 1773:1263–1284