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Abstract

The change of cell polarity is usually associated with invasion and metastasis. Partial reverse cell polarity in IDC-NOS may play a role in lymphatic tumor spread. Rac1 is a kind of polarity related protein. It plays an important role in invasion and metastasis in tumors. We here investigated the expression of Rac1 and partial reverse cell polarity status in breast cancer and evaluated their value for prognosis in breast cancer. The association of the expression of Rac1 and MUC-1 with clinicopathological parameters and prognostic significance was evaluated in 162 cases of IDC-NOS paraffin-embedded tissues by immunohistochemical method. The Rac1 messenger RNA expression was measured by real-time polymerase chain reaction in 30 breast cancer patients, which was divided into two groups of partial reverse cell polarity and no partial reverse cell polarity. We found that lymph node metastasis of partial reverse cell polarity patients was higher than no partial reverse cell polarity patients (Z = −4.030, p = 0.000). Rac1 was upregulated in partial reverse cell polarity group than no partial reverse cell polarity group (Z = −3.164, p = 0.002), and there was correlationship between the expression of Rac1 and partial reverse cell polarity status (r_s = 0.249, p = 0.001). The level of Rac1 messenger RNA expression in partial reverse cell polarity group was significantly higher compared to no partial reverse cell polarity group (t = −2.527, p = 0.017). Overexpression of Rac1 and partial reverse cell polarity correlates with poor prognosis of IDC-NOS patients (p = 0.011). Partial reverse cell polarity and lymph node metastasis remained as independent predictors for poor disease-free survival of IDC-NOS (p = 0.023, p = 0.046). Our study suggests that partial reverse cell polarity may lead to poor prognosis of breast cancer. Overexpression of Rac1 may lead to polarity change in IDC-NOS of the breast. Therefore, Rac1 could be a therapeutic target for breast cancer.

Keywords

Breast cancer polarity partial reverse cell polarity Rac1 prognosis

Introduction

Cell polarity refers to the asymmetry of cell morphology, distribution of protein, and cell function. It is required in cell development, maintaining apical–basal polarity, damage repair, and other physiological processes.¹ The loss of cell polarity and the disorder of the tissue structure are usually associated with the process of epithelial malignant transformation. When the polarity of tumor cell changes, it is more likely to invade and metastasize.²

Breast cancer is the most common malignant disease among tumors in women. Invasion and metastasis are the main causes of death in breast cancer. Invasive ductal carcinoma, not otherwise specified (IDC-NOS) is the most common histological type, accounting for approximately 40%–75% of all cases.^3,4 The mucin family protein MUC-1 is normally expressed at the apical membrane of mammary gland duct. It is instead localized to the external surface of the cell clusters, which is described as polarity reversal in invasive micropapillary carcinoma (IMPC).⁵ In the daily activities, we have noticed that some IDC-NOS also showing the presence of MUC-1 immunoreactivity at the periphery of some of the tumor cell clusters, similar to that observed in IMPC, suggesting the possibility of the focal presence of reversed cell polarity in these cases. Acs et al.⁶ found that the presence of partial reverse cell polarity (PRCP) in IDC-NOS may play a role in lymphatic tumor spread. However the mechanism of PRCP in IDC-NOS is still unclear.

Until now there are a variety of proteins which are considered to be related to cell polarity, such as Rho family, Par, Scribble, and Crumbs complex. Rac is a member of the Rho family of small GTPases (guanosine triphosphatases). Rac1 has been linked to the induction of epithelial polarity in cells adhering to extracellular matrix.⁷ Increasing evidence suggests that Rac1 plays an important role in cell migration, loss of adhesion, invasion, and metastasis in tumors.⁸ But the expression of Rac1 in IDC-NOS and the relationship between Rac1 and PRCP in IDC-NOS have not been reported. This study was undertaken to evaluate the expression of Rac1 in breast IDC-NOS and to evaluate the potential importance as a prognostic indicator in IDC-NOS patients.

Materials and methods

Patients and tissue samples

Paraffin-embedded materials from 162 patients of IDC-NOS of the breast who underwent surgery in 2006–2009 was provided by the Department of Pathology, Third Central Hospital of Tianjin, Tianjin, China, with the approval of the Tianjin Third Central Hospital Institutional Review Board, and informed consent had been obtained. The histopathology was reviewed, and the diagnosis of each case was confirmed independently by two pathologists using the World Health Organization (WHO) criteria.³ All patients were female and the median age at the diagnosis was 53 years (range: 28–84 years old). The patients were followed up for 1–122 months with a median follow-up time of 79 months.

Clinicopathological features

Tumor size was measured as the largest dimension of the microscopic invasive component in pathologic sections. Immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) was evaluated at the time of diagnosis. Tumors with staining of >1% of tumor cell nuclei were defined as positive for ER and PR.⁹ HER2 immunostaining was classified according to the Dako HercepTest scoring system. All those cancers scoring 2+ were subjected to HER2 gene amplification analysis using fluorescence in situ hybridization (FISH) test. HER2 status was divided into two categories: negative: 0/1+ and 2+ (FISH-negative) and positive: 3+ and 2+ (FISH-positive). Lymph node metastatic status was referred to the postoperative pathologic diagnosis. The tumor grade and tumor–node–metastasis (TNM) stage of the cells were according to the standard of WHO.³

Immunohistochemistry

For all specimens, 4 µm serial sections were prepared from one representative block for each tumor. The sections were deparaffinized and rehydrated with xylene and a series of grades of alcohol. Antigen retrieval was carried out in 5 mM citrate buffer (pH 6.0) for 2.5 min in an autoclave, followed by cooling at room temperature for 45 min. After inactivation of endogenous peroxidase with 3% H₂O₂, sections were blocked with 10% goat serum for 30 min. And then, the sections were incubated with primary antibodies, Rac1 (1:1000; ab33186; Abcam, Cambridge) and MUC-1 (1:500; ab109185; Abcam) for overnight at 4°C. Sections were then reacted with biotinylated antibody and streptavidin–biotin–peroxidase (Zhongshan Golden Bridge, China). Diaminobenzidine was used as a chromogen substrate. Finally, the sections were washed in distilled water and weakly counterstained with hematoxylin. Normal breast samples were used as positive control. Negative controls were prepared omitting the primary antibody.

For Rac1 evaluation, the percentage of positive cells was assessed and classified into three groups:¹⁰ (0) 0%, (1+) 1%−33%, and (2+) ≥34%. For statistical analysis, the results were presented as a high expression (2+) or a low expression (0 and 1+) for tumor cells. PRCP was defined in this study as the presence of tumor cell clusters that were partly but not completely fringed with strong MUC-1 immunohistochemical staining facing the stroma, associated with decreased cytoplasmic staining.¹¹ While whole cytoplasmic membrane staining of MUC-1 was defined as no PRCP.

Real-time polymerase chain reaction

Breast cancer tissues (n = 30) included 15 PRCP and 15 no PRCP patients. At the time of surgery, one piece of breast cancer tissue was embedded in paraffin for immunohistochemistry and an adjacent tumor piece was snap-frozen and stored in liquid nitrogen to be used for real-time polymerase chain reaction (RT-PCR) experiments. Breast cancer tissues were obtained from the Tianjin Third Central Hospital, China. TRIzol reagent was used to isolate RNA from cultured cells. Complementary DNA (cDNA) was synthesized by SuperScript III Reverse Transcriptase (Invitrogen, USA) using random primers following the manufacturer’s protocol. PCR reactions were performed using QuantiTect SYBR Green RT-PCR Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The sequences of the semiquantitative RT-PCR primers are as follows—Rac1: 5′-AGC TTT TGC GGA GAT TTT GA-3′ (forward) and 5′-CCC GTG ACA CTT TCA TTC CT-3′ (reverse), glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′ (forward) and 5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′ (reverse). Initial denaturation was at 94°C for 2 min, followed by 30 cycles of 94°C for 20 s and 60°C for 30 s. The relative amounts of Rac1 transcript were normalized to those of the GAPDH transcript.

Statistical analysis

Statistical analysis was carried out using SPSS 19.0 (SPSS Inc., Woking, UK). Differences between groups were analyzed using Mann–Whitney U test. Correlations between two variables were evaluated by Spearman’s rank correlation test. The t test was used to detect the difference of Rac1 messenger RNA (mRNA) expression level between the PRCP and no PRCP breast cancer tissues. Survival curves for disease-free survival (DFS) and overall survival (OS) were constructed using the method of Kaplan and Meier, and the differences between the two groups were assessed using the log-rank test. Cox proportional hazards models were used to perform univariate and multivariable analyses. A two-tailed p value < 0.05 was considered to be statistically significant.

Results

PRCP correlated with lymph node metastasis of IDC-NOS patients

To verify the proportion of PRCP in IDC-NOS, we used MUC-1 as a marker of polarity. We defined MUC-1 immunohistochemistry staining localized to at least part of the external surface of the cell clusters as PRCP (Figure 1(a)) and the whole cytoplasmic membrane staining of tumor cell clusters was defined as no PRCP (Figure 1(b)). The results showed that among the total of 162 cases of IDC-NOS, 31 (19.1%) cases were with PRCP and 131 (80.9%) were without PRCP. All the patients accepted nodal resection, 79 (48.8%) cases were node-negative and 83 (51.2%) cases were node-positive. In total, 26 (83.9%) showed lymph node metastasis in PRCP group. In contrast, only 57 (43.5%) of the no PRCP cases in which axillary nodes were evaluated had metastasis. Lymph node metastasis was significantly higher in PRCP compared with no PRCP (p = 0.000; Table 1). No significant difference in patient age, tumor size, histological grade, ER status, PR status, and HER2 status was identified between the PRCP and no PRCP group (p > 0.05; Table 1).

Figure 1.

(a) Immunohistochemical staining of MUC-1 in PRCP of IDC-NOS (200×). MUC-1 immunohistochemistry staining localized to at least part of the external surface of the cell clusters. (b) Immunohistochemical staining of MUC-1 in no PRCP of IDC-NOS (200×). MUC-1 immunohistochemistry staining localized to whole cytoplasmic membrane staining of tumor cell clusters. (c and d) Rac1 expression in breast cancer patients by immunohistochemistry, which was mainly expressed in nucleus and cytoplasm.

Table 1.

Clinicopathological characteristics of PRCP and no PRCP patients.

Characteristics	PRCP (%)	no PRCP (%)	Z	p value*
Age (years)
≤53	16 (51.6)	69 (52.7)	−0.106	0.916
>53	15 (48.4)	62 (47.3)
Tumor size (T stage)
T1	8 (25.8)	50 (38.2)	−0.859	0.390
T2	22 (71.0)	71 (54.2)
T3–T4	1 (3.2)	10 (7.6)
Histological grade
I	2 (6.5)	15 (11.5)
II	24 (77.4)	98 (74.8)	−0.737	0.461
III	5 (16.1)	18 (13.7)
Lymph node status
No metastasis	5 (16.1)	74 (56.5)	−4.030	0.000
metastasis	26 (83.9)	57 (43.5)
ER status
Negative	17 (54.8)	49 (37.4)	−1.771	0.077
Positive	14 (45.2)	82 (62.6)
PR status
Negative	10 (32.3)	50 (38.2)	−0.611	0.541
Positive	21 (67.7)	81 (61.8)
HER2 status
Negative	19 (61.3)	99 (75.6)	−1.603	0.109
Positive	12 (38.7)	32 (23.7)
Rac1 expression
Low	3 (9.7)	52 (39.7)	−3.164	0.002
High	28 (90.3)	79 (60.3)

PRCP: partial reverse cell polarity; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2.

p values were calculated by Mann–Whitney U test.

Elevated Rac1 expression in IDC-NOS correlated with PRCP and lymph node metastasis

In order to verify the expression of Rac1 in breast cancer, we detected the expression of Rac1 in breast cancer patients by immunohistochemistry. The results showed that Rac1 was mainly expressed in nucleus and cytoplasm and was shown as brown particles (Figure 1(c) and (d)). In the 162 IDC-NOS samples examined, 107 (66.0%) showed high expression of Rac1, in which there were 28 (90.3%) cases with PRCP and 79 (60.3%) cases without PRCP. The expression of Rac1 in PRCP group was significantly higher than that in no PRCP group (Z = −3.164, p = 0.002; Table 1).

In addition, through Spearman’s rank correlation analysis, we found that the expression of Rac1 in IDC-NOS was positively correlated with lymph node metastasis (r_s = 0.370, p = 0.000) and PRCP status (r_s = −0.249, p = 0.001), but no significant association was observed between the expression of Rac1 and age, tumor size, histological grade, ER, PR, and HER2 status (Table 2). It means that Rac1 was upregulated in PRCP group than no PRCP group, and there was correlationship between the expression of Rac1 and PRCP status.

Table 2.

Rac1 expression and clinicopathological parameters of IDC-NOS patients.

Characteristics	Rac1 expression		r_s	p value*
Characteristics	Low	High	r_s	p value*
Age (years)
≤53	30	55	0.030	0.707
>53	25	52
Tumor size (T stage)
T1	25	33	0.123	0.118
T2	26	67
T3–T4	4	7
Histological grade
I	7	10
II	42	80	0.080	0.311
III	6	17
Lymph node status
No metastasis	41	38	0.370	0.000
Metastasis	14	69
ER status
Negative	24	42	0.042	0.593
Positive	31	65
PR status
Negative	19	41	−0.037	0.640
Positive	36	66
HER2 status
Negative	38	80	−0.060	0.445
Positive	17	27
MUC-1 expression
PRCP	3	28	0.249	0.001
No PRCP	52	79

IDC-NOS: invasive ductal carcinoma, not otherwise specified; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2; PRCP: partial reverse cell polarity.

p values were calculated by Spearman’s rank correlation test.

Rac1 mRNA is upregulated in IDC-NOS with PRCP than no PRCP

To further explore whether Rac1 was upregulated in PRCP group, we examined the expression of Rac1 mRNA in 30 breast cancer patients by RT-PCR. Total RNA from PRCP (n = 15) and no PRCP (n = 15) breast cancer tissues was analyzed. We found that the mRNA expression of Rac1 was significantly higher in PRCP group than no PRCP group (t = −2.527, p = 0.017; Figure 2).

Figure 2.

The expression of Rac1 mRNA in 15 PRCP patients and 15 no PRCP patients. Data obtained by real-time PCR amplification of mRNA are plotted. mRNA is normalized by GAPDH. ΔCt = Ct_mRNA − Ct_GAPDH. The boxes represent median with interquartile range, whiskers go to the highest or lowest point. p values were obtained applying the independent sample t test.

Overexpression of Rac1 and PRCP correlated with poor prognosis of IDC-NOS patients

An important question was whether Rac1 expression clinically correlated with prognosis of IDC-NOS. To address this issue, we performed a survival analysis of DFS and OS in all breast cancer patients. Kaplan–Meier analysis revealed that the DFS of patients with high expression of Rac1 was significantly shorter than those with low Rac1 expression in IDC-NOS (p = 0.011; Figure 3(a)). But there was no significant correlation between Rac1 expression and the OS of IDC-NOS (p > 0.05). To further evaluate whether PRCP expression can predict prognosis, we performed a survival analysis of DFS and OS in all IDC-NOS patients. Kaplan–Meier survival curves showed that the DFS of patients with PRCP was significantly shorter than no PRCP patients in IDC-NOS (p = 0.000; Figure 3(b)). But there was no significant correlation between PRCP status and the OS of IDC-NOS (p > 0.05). A unitivariate Cox regression analysis confirmed that tumor size, lymph node status, Rac1 expression, and PRCP predicted an increase in risk for poor DFS of patients with IDC-NOS (p < 0.05; Table 3). But the multivariate Cox regression analysis showed that only PRCP (p = 0.023) and lymph node metastasis (p = 0.046) remained as independent predictors for poor DFS of IDC-NOS (Table 3).

Figure 3.

Kaplan–Meier curves showing the predictive value of Rac1 expression and PRCP status in IDC-NOS. (a) DFS of patients with high expression of Rac1 was significantly shorter than those with low Rac1 expression in IDC-NOS (p = 0.011). (b) DFS of patients with PRCP was significantly shorter than no PRCP patients in IDC-NOS (p = 0.000).

Table 3.

Univariate and multivariate Cox regression analysis for disease-free survival of IDC-NOS patients.

Variables	Univariable			Multivariable
Variables	HR	95% CI	p value	HR	95% CI	p value
Age (years)
≤53 vs >53	1.067	0.565–2.015	0.842	–	–	–
Tumor size (T stage)
T1 vs T2 vs T3–T4	1.969	1.113–3.484	0.020	1.548	0.843–2.845	0.159
Histological grade
I vs II vs III	1.420	0.746–2.703	0.286	–	–	–
Lymph node status
No metastasis vs metastasis	3.727	1.763–7.879	0.001	2.316	1.016–5.279	0.046
ER status
Negative vs positive	0.597	0.316–1.130	0.113	–	–	–
PR status
Negative vs positive	0.770	0.400–1.482	0.435	–	–	–
HER2 status
Negative vs positive	0.604	0.277–1.319	0.206	–	–	–
Rac1 expression
Low vs high	2.963	1.236–7.103	0.015	1.670	0.660–4.222	0.279
MUC-1 expression
PRCP vs no PRCP	3.258	1.698–6.253	0.000	2.190	1.113–4.308	0.023

IDC-NOS: invasive ductal carcinoma, not otherwise specified; HR: hazard ratio; CI: confidence interval; ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2; PRCP: partial reverse cell polarity.

Discussion

Breast cancer is one of the high incidence malignant tumors in female. Invasion and metastasis are the most important factors that affect the outcome and prognosis of patients with breast cancer. But the mechanisms of invasion and metastasis are very complex. In this study, we mainly talk about cell polarity. The internal structure and protein components of normal epithelial cells are asymmetrically distributed, so that the cell can perform many important physiological functions, such as secretion, absorption, barrier formation, and so on.¹² When the polarity of tumor cells changes, it is more likely to invade and metastasize. Previous literature has proved that PRCP was related to lymph node metastasis in breast cancer.^6,13 In this study, we also found that lymph node metastasis was significantly higher in PRCP patients compared with no PRCP patients. Meanwhile, PRCP was significantly correlated with the poor DFS of IDC-NOS. But we found that polarity reversal was not related with OS of IDC-NOS. This may be due to the prognosis of breast cancer patients is usually good, and our follow-up time is relatively short.

MUC-1 is a high molecular weight, heavily glycosylated transmembrane glycoprotein, expressed on the apical surface of a wide variety of epithelial cells.⁵ In vitro studies showed that increased MUC-1 expression results in decreased adhesion between adjacent cells and between epithelial cells and extracellular matrix.¹⁴ The polarity reversal contributes to the distribution of MUC-1, which is secreted by the apical of the glands to the outer surface of the cell clusters.⁵ In this study, our results demonstrated that PRCP in IDC-NOS was positively correlated with lymph node metastasis (r_s = 0.318, p = 0.000). Thus, we concluded that IDC-NOS patients with PRCP were more likely to undergo invasion and metastasis. But the mechanism of PRCP is still unclear.

The polarity of epithelial cells is regulated by polarity associated proteins, such as Rho GTPases, Par, Scribble, and Crumbs complexes. They are mutually regulated and interconnected with small Rho GTPase signaling through recruiting specific guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) for Rho GTPases. Ultimately, they spatially control the actin cytoskeleton to promote the establishment and maintenance of cell–cell junctions and polarized phenotype.¹⁵ Recently, they have also been shown to contribute to the regulation of malignant progression, including invasion and metastasis.^16,17 Rac is a member of the Rho GTPases. Increasing evidence suggests that Rac1 plays an important role in cell migration, loss of adhesion, invasion, and metastasis in tumors.^8,18 Rozenchan et al.¹⁰ found that Rac1 was upregulated in carcinoma-associated fibroblasts of breast cancer and was associated with lymph node metastasis. But the expression of Rac1 in IDC-NOS of breast and the relationship between the expression of Rac1 and PRCP have not been reported. Our results showed that the expression of Rac1 was upregulated in IDC-NOS with PRCP than no PRCP group. In addition, the expression of Rac1 was positively correlated with lymph node metastasis and PRCP in IDC-NOS.

In this study, the expression of Rac1 was significantly correlated with the poor DFS of IDC-NOS (p = 0.011). This may indicate that Rac1 status could be helpful to evaluate the risk of tumor recurrence and metastasis of IDC-NOS patients. It was also reported that Rac1 was upregulated in hepatocellular carcinoma. Moreover, survival analysis indicated that hepatocellular carcinoma patients with strong Rac1 expression had shorter disease-specific survival than those with weak expression. Multivariate analysis also showed that Rac1 overexpression could be a predictor of poor prognosis.¹⁹

Therefore, a better understanding of the molecular mechanisms of the regulatory relationships between Rac1 and PRCP is critical for the development of efficacious treatments for breast cancer. Activation of Rac1 and Cdc42 by mPar6 mPar3/mPar6/aPKC protein complex and interaction with each other are required in the formation of polarity.¹⁵ Other literatures reported that Rac1 could activate downstream PAK1, which could further activate the PAK1/LIMK1/cofilin1 pathway, leading to the occurrence and invasion of tumor.²⁰ Archibald et al.²¹ reported that oncogenic suppression of apoptosis uncovers a proliferative role for Par3 loss by activating a Tiam1/Rac1/C-jun n-terminal kinase (JNK)-dependent pathway that contributes to mammary tumor growth. Downregulation of Par3 in breast cancer cells affects Tiam1 localization, which leads to Rac1 activation, diminished E-cadherin stability, loosening of adherens junctions, and finally increased cell invasion and metastasis.²² Rac1 was shown to be essential for the tumor formation. It was also shown that this was via promoting Rac1, Erk-dependent hyperproliferation through a Rac1/PAK1/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) pathway.²³

In conclusion, these results indicate that overexpression of Rac1 may lead to PRCP in IDC-NOS of the breast. In addition, the histological features of PRCP were associated with higher lymph node metastasis and poor prognosis of breast IDC-NOS. So PRCP in IDC-NOS should be taken seriously in our daily diagnosis. These findings may be useful in targeted therapies for patients with breast cancer. Association between Rac1 expression and PRCP in IDC-NOS needs to be better understood, and further studies are needed to clarify the molecular basis involved in this process.

Footnotes

Acknowledgements

B.L. and J.X. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

The study protocol was approved by the ethics committee of the Third Central Hospital of Tianjin, China, and written informed consent for the utilization of clinical data was obtained from all the enrolled patients.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Third Central Hospital National Natural Science Foundation Incubation Project, Tianjin, China (No. 2017YNY2 and 2017YNR3), Key Research Project of Tianjin Healthy Bureau (No. 07KG14), the National Natural Science Foundation of China (No. 81301985), and the Natural Science Foundation of Tianjin (No. 14JCQNJC14000).

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High expression of Rac1 is correlated with partial reversed cell polarity and poor prognosis in invasive ductal carcinoma of the breast