Original Article Clinicopathological significance of PTEN and PI3K/AKT signal transduction pathway in non-small cell lung cancer

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Int J Clin Exp Pathol 2013;6(10):2112-2120 www.ijcep.com /ISSN:1936-2625/IJCEP1308017

Original Article

Clinicopathological significance of PTEN and

PI3K/AKT signal transduction pathway

in non-small cell lung cancer

Fen Yun1_{, Yongfeng Jia}1_{, Xiuxia Li}1_{, Li Yuan}2_{, Qinnuan Sun}1_{, Huiling Yu}1_{, Lin Shi}1_{, Hongwei Yuan}1

1_{Department of Pathology, The First Affiliated Hospital of Inner Mongolia Medical University, Huhhot, 010059,} China; 2_{Shandong Heze Medical College, Heze, Shandong 274000}

Received August 8, 2013; Accepted September 3, 2013; Epub September 15, 2013; Published October 1, 2013

Abstract: A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not

un-derstood fully. The purpose of our study was to examine the impact of EGFR, TGF-α, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-α and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expres

-sion by in situ hybridization. There were progressive loss of PTEN expres-sion and significant increasing in EGFR, TGF-α, P-AKT expression from benign samples to NSCLC (p<0.05). The overexpression of EGFR, TGF-α, P-AKT and

loss of PTEN expression were correlated to differentiation extent of cancer tissue, metastasis of lymph nodes and

histological classification. Thus, alteration of EGFR, TGF-α, P-AKT and PTEN are likely important molecular events in

pathogenesis and carcinogenesis of NSCLC.

Keywords: Non-small cell lung cancer (NSCLC), P-AKT, TGF-α, EGFR, PTEN

Introduction

Non-small Cell Lung Cancer (NSCLC) is one of the most formidable health problems in terms of morbidity and mortality facing the mankind today. It is the most common cancer worldwide accounting for 80% of all lung cancer in men and women. Premalignant lesions of tissue show features of epithelial, moderate and severe epithelial dysplasia carry the highest risk for malignant transformation. Identification of high risk premalignant lesions with increased susceptibility to NSCLC and consequent aggressive follow up for early detection and treatment may help in down staging of the can-cer and better prognosis [1]. Human NSCLCs show a variety of genetic changes, with differ-ent changes in differdiffer-ent tumors [2]. PTEN (phosphatase and tensin homolog deleted on chromosome TEN), is a tumor suppressor gene mutated in a variety of human cancers includ-ing prostate, breast, brain, endometrial, glio-blastoma, and melanoma [3-6]. PTEN

expres-sion has been down regulated in many malignancies including lung carcinoma [7], oral carcinoma colorectal adenocarcinoma [8, 9], breast cancer, colon cancer, and renal cell car-cinoma [10, 11].

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PTEN and PI3KAKT pathway in non-small cell lung cancer

Figure 1. PTEN showing cytoplasmic expression in NSCLC and benign samples of lung (A: Positive sion of PTEN in lung normal tissue; B: Positive expres-sion of PTEN in lung Squamous carcinoma tissue; C: Positive expression of PTEN in lung adenocarcinoma

[image:2.612.95.517.73.391.2]

tissue. Original magnification ×400).

Table 1.Loss of PTEN expression in NSCLC and benign samples

Tissue PTEN X2 _P_-value*

+ - Positive ratio (%)

NSCLC 22 44 33.30% 6.083 0.014

Benign sample 8 2 80%

Note: *_{Chi-square test.}

Table 2.Correlation between PTEN and clinicopathological factors

Factors PTEN X2 _P_-value*

- +~+++

Age

≤53 12 5 0.158 0.691

>53 32 17

Sex

Male 32 18 0.66 0.417

Female 12 4

Lymph node metastasis

Positive 28 7 5.961 0.015

Negative 16 15

Pathological pattern

adenocarcinoma 20 4 4.714 0.03

Squamous carcinoma 24 18

Differentiation grade

Moderate-well 24 20 8.727 0.003

Poor 20 2

This study attempts to study the differential expression pat-tern of EGFR, TGF-α and P-AKT and PTEN protein for its rele-vance in development and pro-gression of NSCLC.

Materials and methods

Study population

[image:2.612.89.374.430.476.2] [image:2.612.91.369.517.713.2]

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Histopathologic-PTEN and PI3KAKT pathway in non-small cell lung cancer

ally, the NSCLCs were cate-gorized as well differentiat-ed and moderately differentiated -44 cases and poorly differentiated -22 cases, 35 cases with and 31 cases without lymph node metastasis. The benign samples of lung (males and females), 31-65 years of age (median age, 48 years) were taken as control.

The protocol of this study was approved by the Protocol Review Committee and the Bioethics Commi- ttee of Inner Mongolia Medical University.

PTEN in situ hybridization

In order to detect PTEN expression, in situ hybrid-ization assays were

per-Figure 2. EGFR expression in NSCLC and benign samples of lung (A: Negative expression of EGFR in lung normal tissue; B: Positive expression of EGFR in Squamous car-cinoma tissue; C: Positive expression of EGFR in lung

[image:3.612.96.519.74.392.2]

ad-enocarcinoma tissue. Original magnification ×400).

Table 3. EGFR expression in NSCLC and benign samples

Tissue EGFR X2 _P_-value*

NSCLC 46 20 69.70% 4.367 0.037

Benign sample 3 7 30.00%

Table 4. Correlation between EGFR and clinicopathological factors

Factors EGFR X2 _P_-value*

- +~+++

Age

≤53 6 11 0.27 0.603

>53 14 35

Sex

Male 16 34 0.047 0.828

Female 4 12

Positive 4 31 12.569 0.001

Negative 16 15

Moderate-well 18 26 7.03 0.008

Poor 2 20

[image:3.612.90.389.427.476.2] [image:3.612.91.388.515.713.2]

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PTEN and PI3KAKT pathway in non-small cell lung cancer

formed on serial 5 microns thick sections from the orig -inal blocks and the TMA block, using a kit for in situ hybridization (MK1276, Bo-ster Biotech Co, Wuhan, China), according to the provider’s specifications as described in detail else-where. The poly-A probe, used as an indicator of the preservation of mRNA in the cells (positive control), resulted mainly in dark brown nuclear staining and less in a cytoplasmic one.

Immunohistochemical

analysis

[image:4.612.94.519.74.393.2]

Formalin fixed paraffin-embedded tissue blocks were cut in 5 microns thick serial sections. The sec-tions were deparaffinized, rehydrated and rinsed in

[image:4.612.91.389.432.477.2]

Figure 3. TGF-α expression in NSCLC and benign samples of lung (A: Negative expression of TGF-α in lung normal tissue; B: Positive expression of TGF-α in lung Squamous carcinoma tissue; C: Positive expression of TGF-α in lung adenocarcinoma tissue. Original magnification ×400).

Table 5. TGF-α expression in cytoplasm of NSCLC and benign samples

Tissue TGF-α X2 _P_-value*

NSCLC 47 19 71.20% 4.850 0.028

Table 6.Correlation between TGF-α and clinicopathological factors

Factors TGF-α X2 _P_-value*

- +~+++

Age

≤53 6 11 0.142 0.706

>53 13 36

Sex

Male 14 36 0 1

Female 5 11

Positive 5 30 7.645 0.006

Negative 14 17

Moderate-well 16 28 3.695 0.055

Poor 3 19

[image:4.612.92.390.518.714.2]

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PTEN and PI3KAKT pathway in non-small cell lung cancer

[image:5.612.94.519.74.390.2]

phosphate buffer saline (PBS). An Immunohistoche-mical assay for EGFR, TGF-α, P-AKT was performed on consecutive paraffin sec -tions using streptavidin-bio-tin method. Monoclonal mouse antihuman EGFR antibody (ZM0093, Beijing ZSGB Company, China), monoclonal rabbit antihu-man TGF-α antibody (ZA2-54, Beijing ZSGB Company, China) and monoclonal mouse antihuman P-AKT antibody (sc16646, Beijing ZSGB Company, China) were used as primary anti-bodies respectively. After antigen retrieval slides were incubated with primary anti-body, followed by second-ary biotinylated antibody. Sections were washed in PBS and then incubated

Table 7. P-AKT expression in cytoplasm of NSCLC and benign samples

Tissue P-AKT X2 _P_-value*

NSCLC 50 16 75.80% 10.048 0.002

Table 8. Correlation between P-AKT and clinicopathological factors

Factors P-AKT X2 _P_-value*

- +~+++

Age

≤53 3 14 0.166 0.683

>53 13 36

Sex

Male 12 38 0 1

Female 4 12

Positive 4 31 6.662 0.010

Negative 12 19

Moderate-well 14 30 4.125 0.042

Poor 2 20

[image:5.612.91.389.432.478.2]

Figure 4. P-AKT immunostaining showing expression in

cytoplasm of NSCLC and benign samples of lung (A:

Neg-ative expression of P-AKT in lung normal tissue; B: Posi

-tive expression of P-AKT in lung Squamous carcinoma tissue; C: Positive expression of P-AKT in lung adenocar

[image:5.612.92.387.518.713.2]

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[image:6.612.90.344.84.139.2]

PTEN and PI3KAKT pathway in non-small cell lung cancer

Table 9. Correlation between EGFR and TGF-α expression

EGFR TGF-α X2 _P_-value*

+

-+ 37 9 0.309 0.012

- 10 10

[image:6.612.91.344.188.240.2]

Table 10. Correlation between EGFR and P-AKT expression

EGFR P-AKT X2 _P_-value*

+

-+ 39 7 0.319 0.022

- 11 9

[image:6.612.91.344.290.345.2]

Table 11. Correlation between P-AKT and TGF-α expression

P-AKT TGF-α X2 _P_-value*

+

-+ 41 9 0.421 0.002

- 6 10

with streptavidin peroxidase. Finally chromo-gen Diaminobenzidine (DAB) was used and sec-tion were counterstain with hematoxylin.

Table 12. Correlation between EGFR and PTEN expression

EGFR PTEN X2 _P_-value*

+

-+ 9 37 -0.443 0.001

- 13 7

Table 13. Correlation between TGF-α and PTEN expression

TGF-α PTEN X2 _P_-value*

+

-+ 11 36 -0.331 0.007

- 11 8

Table 14. Correlation between P-AKT and PTEN expression

P-AKT PTEN X2 _P_-value*

+

-+ 12 38 -0.35 0.004

- 10 6

Scoring method

Tumors were classified as EGFR, TGF-α, P-AKT and PTEN negative (i.e. low expression) if less than 10% of cells displayed positivity. If equal to or greater than 10% of cells were posi-tive for EGFR, TGF-α, P-AKT and PTEN (i.e. high expression) were considered as positive.

A total of 5-6 fields from each tissue section were chosen, and 100 cells from each field were counted at final magnification at 400X. With every batch of staining a positive and nega-tive control were used to verify the standard of staining.

The percentage of EGFR, TGF-α, P-AKT and PTEN positive cells was calculat-ed independently by two pathologists. The correlations between EGFR, TGF-α, P-AKT and PTEN expression and histological grading were studied. Statistical analysis

Chi-square (X)2_{test was performed to}

find out the possible correlation among EGFR, TGF-α, P-AKT and PTEN and other clinical parameters in NSCLC and benign samples of lung tissue. We used the SPSS ver. 14.0 (SPSS Inc., Chicago, IL, USA), with P<0.05 being considered as statisti -cally meaningful.

Results

Loss of PTEN expression in NSCLC

[image:6.612.90.344.393.448.2] [image:6.612.91.344.496.547.2] [image:6.612.91.344.596.649.2]

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PTEN and PI3KAKT pathway in non-small cell lung cancer

speculate that tumor prognostic features cor-related with EGFR, TGF-α P-AKT and PTEN expression in NSCLC.

Discussion

Lung cancers make up 14% of all newly diag -nosed cancers in the USA today, whereas. Though there are many studies on the etiology of cancer but the exact pathogenesis still remains uncertain. The major etiologic factor in the development of lung cancer is smoking, besides, cultural, and genetic differences, hor-monal and possibly infectious factors may also play etiologic roles [18]. The incidence of lung cancer might be due to the cumulative effects of long time exposures to carcinogens, the fail-ings of DNA repair mechanisms and aging [19]. The identification of prognostic and predictive markers is clinically important, because lung cancer is a group of heterogenous diseases with various biological and clinical characteris-tics. Loss of PTEN function and/or overexpres-sion of growth factors and malfunctions in their signal pathways occur frequently in cancers. EGFR, TGF-α and P-AKT have been studied as predictive markers and prognostic factors in prostate, oral, breast and lung cancers [20, 21]. Earlier investigator showed that expres-sion status of EGFR, TGF-α, P-AKT or PTEN in lung cancer. However, to our knowledge, this is the first study to report that EGFR, TGF-α, and P-AKT expression is inversely correlated with PTEN loss in NSCLC. This observation shows that a mechanism by which EGFR, TGF-α, and P-AKT are upregulated in the development and progression of NSCLC. Such a mechanism would include loss of PTEN activity. The upregu-lation of EGFR, TGF-α, and P-AKT might reduce the apoptotic induction thereby contributing to the tumorigenesis. The present study reveals that loss of PTEN expression in NSCLC clinical specimens is significantly correlated with EGFR, TGF-α, and P-AKT overexpression. The study also shows that loss of PTEN expression and EGFR, TGF-α, P-AKT overexpression are signifi -cantly correlated with NSCLC staging, suggest-ing that both of the activities may play an important role in NSCLC development and pro-gression. Earlier investigator showed that loss of PTEN increased remarkably according to dis -ease stage and lymph node metastasis [22, 23]. Our study showing that PTEN gene altera-tion is associated with advanced stage and lymph node involvement, histological classifi

-cation and poor differentiation (Table 2).

Immunohistochemical detection of EGFR

protein

Nuclear immuno reaction for EGFR was consid-ered positive and found in 46/66 cases (69.7%) of NSCLC and 3/10 of benign samples of lung (Figure 2). The result reveals the significant dif -ference between the normal tissue (30%) and cancerous tissue (69.7%) for the EGFR protein (p<0.05). With the progression in lymph node involvement and tumor grade, the rate of EGFR expression significantly increased (Tables 3

and 4).

Immunohistochemical detection of TGF-α

protein

Cytoplasmic and nuclear immuno-reaction for TGF-α was considered positive and found in 47/66 cases (71.2%) of NSCLC and 3/10 of benign samples of lung (Figure 3). In the NSCLC，the overexpression of TGF-α had sig -nificant correlation with the metastasis of lymph nodes (Tables 5 and 6).

Immunohistochemical detection of P-AKT

protein

Nuclear immuno reaction for P-AKT was consid -ered positive and found in 50/66 cases (75.8%) of lung carcinoma and 2/10 of benign samples of lung (Figure 4). The result reveals the signifi -cant difference between the normal tissue (20%) and cancerous tissue (75.8%) for the P-AKT protein (p<0.05). With the progression in lymph node involvement and tumor grade, the rate of P-AKT expression significantly increased (Tables 7 and 8).

The relationship between EGFR, TGF-α, P-AKT and PTEN expression

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PTEN and PI3KAKT pathway in non-small cell lung cancer

lymph node metastasis. Our results suggest that PTEN may play an important role in the regulation of tumor progression and metasta-sis during the development of NSCLC. But the exact role of PTEN in the genesis of NSCLC remains unknown. Studies on PTEN in NSCLC showed frequent genetic alterations and loss of expression but still there are a lot of discrepan-cies toward the drawing of the final conclusion [24]. Recent study demonstrates that loss of PTEN was associated to up-regulation of the EGFR gene, thus contributing to survival of can-cer cells in lung and breast cancan-cers [25, 26]. Our results also showed the strong association of loss PTEN expression with not only EGFR, but also TGF-α, and P-AKT positive expression (p<0.05) in NSCLC.

Acknowledgements

This project was supported by science fund for young scholars of Inner Mongolia Medical University (NY2007QN007).

Disclosure of conflict of interest

There is no conflict of interest among all the authors in this study.

Address correspondence to: Dr. Yongfeng Jia,

Department of Pathology, The First Affiliated

Hospital of Inner Mongolia Medical University, NO.1, Northern Tongdao Street, Huhhot, 010059, China. E-mail: yfjia0479@163.com

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