J Mol Neurosci (2016) 58:137–144 DOI 10.1007/s12031-015-0698-z

The Clinical Utility of TIMP3 Expression in ACTH-Secreting Pituitary Tumor Bowen Sun 1 & Xiaohai Liu 1 & Yakun Yang 1 & Congxin Dai 1 & Ying Li 2 & Yonghui Jiao 1 & Zhenqing Wei 1 & Yong Yao 1 & Ming Feng 1 & Xinjie Bao 1 & Kan Deng 1 & Ning Wang 2 & Renzhi Wang 1

Received: 16 October 2015 / Accepted: 1 December 2015 / Published online: 16 December 2015 # Springer Science+Business Media New York 2015

Abstract In recent years, the tissue inhibitor of metalloproteinase-3 (TIMP3) plays a pivotal role in tumorigenesis, while the role of TIMP3 in adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas remains unclear. In this study, 86 sporadic pituitary tumor specimens, including ACTH (40), GH (18), PRL-secreting (8), and nonfunctioining (20) and non-tumorous pituitary samples (n= 10) were available, and then, the mRNA and protein expression of TIMP3 was quantified by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and immunohistochemistry, respectively. Our findings showed that TIMP3 expression was significantly correlated with Ki-67 expression and the invasiveness of pituitary adenomas. TIMP3 mRNA and protein expression were reduced in ACTH-secreting pituitary adenomas and the other three types of pituitary adenomas compared to adjacent nontumorous pituitary tissues (all p<.01). On the other hand, the expression of TIMP3 was negatively correlated with tumor size and Ki-67 in ACTH-secreting pituitary adenomas. TIMP3 mRNA expression was significantly lower in invasive pituitary adenomas than that in noninvasive ones (1.92-fold, p<.05). TIMP3 protein levels were also significantly lower in the majority of invasive adenomas (1.41-fold, p < .05) Furthermore, TIMP3 mRNA and protein expression were Bowen Sun and Xiaohai Liu contributed equally to this work. * Renzhi Wang [email protected]

1

Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100730, China

2

Department of Neurosurgery, the First Affiliated Hospital, Harbin Medical University, Harbin 150001, China

significantly lower in pituitary giant adenomas than those in microadenomas (2.58-fold, p<.05). In conclusion, the expression of TIMP3 is low in pituitary adenomas including ACTHsecreting pituitary adenomas and negatively associated with tumor aggressiveness. TIMPs may play a potential role in the progression of ACTH-secreting pituitary adenomas and be useful as a biomarker of invasiveness. Keyword TIMP3 . Ki-67 . Clinicopathology . ACTH-secreting pituitary tumor

Introduction It has been reported that most of adrenocorticotrophic hormone (ACTH)-dependent Cushing syndromes are attributed to ACTH-secreting adenomas of the pituitary gland (Cushing 1932). Despite that the size in the majorities of ACTHsecreting tumors is less than 10 mm (Mampalam et al. 1988; Jehle et al. 2008), some tumors are still evolving. Till now, patients with Cushing disease are accustomed to undergoing transsphenoidal surgery, because this way induces remission in 70–90 % of patients. However, patients with large ACTHsecreting adenomas can obtain low remission rate (Petersenn et al. 2015). Thus, it is essential to elucidate the pathogenesis and molecular markers in the aggressiveness of ACTHsecreting pituitary adenomas. Tissue inhibitor of metalloproteinase-3 (TIMP3) acts as a putative tumor suppressor gene and is located on chromosome 22q12.3 (Barski et al. 2010). TIMP3 interacts with the extracellular matrix and antagonizes the activity of matrix metalloproteinases (MMPs) with high affinity. As reported, the reduced expression of TIMP3 contributes to tumor growth and invasion by increasing MMPs activity in the extra cellularmatrix (Pavloff et al. 1992; Yu et al. 2000; Lee et al.

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2007). At the same time, decreased TIMP3 expression was associated with poor prognosis in various tumors (Darnton et al. 2005; Smith et al. 2008; De Schutter et al. 2009; Catasus et al. 2013). However, TIMP3 plays an important role in cancer progression, including metastasis (Qi et al. 2003), apoptosis (Mahller et al. 2008), drug sensitivity in prostate cancer cell lines (Shinojima et al. 2012), and inhibition of tumor growth in lung cancer (Chetty et al. 2008). To date, few report identified TIMP expressions in head and neck cancer and thyroid cancer (Kornfeld et al. 2011; Anania et al. 2011). However, the role of TIMP3 in the pathogenesis of ACTH-secreting adenomas is not known. In this study, we investigated the expression of TIMP3 in human ACTH-, GH, and PRL-secreting, and non-functioning pituitary tumors and paratumoral normal pituitary tissues, and evaluated its clinicopathologic values.

Materials and Methods Patients and Samples Eighty-six sporadic pituitary tumor tissues (40 ACTH-, 18 GH-, 8 PRL-secreting, and, 20 non-functioining,) and 10 adjacent non-tumorous pituitary tissues (ANPTs) were obtained from patients at Peking Union Medical College Hospital (26 men, 70 women, aged 8–68 years with a mean age of 40.3 years) who underwent transsphenoidal surgery between January 2014 and August 2014. All of the surgical specimens were frozen in liquid nitrogen and stored at −80 °C (clinical data were retrieved from medical files). We performed selective adenomectomy plus adequate resection of adjacent nontumorous pituitary tissues in patients in the ANPT group, this surgical method was based upon our previous study (Yang et al. 2014). Tables 1 and 2 show the clinical data and characteristics of 40 patients with Cushing’s disease. The diagnosis of biological behavior was made according to preoperative magnetic resonance imaging (MRI)/computed tomography, gross observation at surgery, or postoperative histopathologic findings. Invasive adenomas were defined as fulfilling one of three conditions: (1) Knosp classification grade III and IV; (2) infiltration of the bones and cavernous sinus or encasement of sinus structures observed during operation; and (3) dural invasion as determined by microscopic histopathology. There were 24 invasive adenomas and 62 noninvasive adenomas. We classified pituitary adenomas into microadenomas (tumor diameter <1 cm, 29 cases), macroadenomas (tumor diameter 1–3 cm, 45 cases), and giant adenomas (tumor diameter >3 cm, 12 cases) as determined by computed tomography or MRI. The study was approved by the Ethical Committee of Peking union medical college hospital, and all patients had written informed consent.

J Mol Neurosci (2016) 58:137–144

Total RNA Extraction and Reverse Transcription Pituitary adenoma tissues and ANPTs from 86 patients obtained during transsphenoidal surgery were frozen in liquid nitrogen and stored at −70 °C (−80 or −70) until used. Total RNA was extracted and isolated with Trizol reagent (Invitrogen). The quantity of RNA were assessed by NanoDrop 2000. Complementary DNA (cDNA) was synthesized with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) in 20 μl reaction volumes with random primers. Real-Time Quantitative PCR Analysis PCR reactions were amplified for 35 cycles with an annealing temperature of 55 °C. Data were normalized to GAPDH levels. The primers used for RT-PCR were as follows: TIMP3 forward:5′-GTCTATGATGGCAAGATGTAC-3′ TIMP3 reverse: 5′-AAGCAAGGCAGGTAGTAGC-3′ GAPDH forward:5′-GGTGGTCTCCTCTGACTT CAACA-3′ GAPDH reverse: 5′-GTTGCTGTAGCCAAATTCG TTGT-3′

Western Blot Analysis Total tissue protein was extracted with Trizol (Invitrogen) according to the manufacturer’s protocol, separated by 12 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membrane (NC), and followed by incubation with primary antibody (anti-GAPDH:1:2000, epitomic, #5632-1; anti-TIMP3:1:1000, Cell Signaling, #5673) overnight at 4 °C. Membranes were subsequently incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature, developed using ECL Western Blotting Detection Reagents (Millipore), and visualized by Kodak film exposure detection system. Films were scanned and quantified by using Quantity one (Bio-Rad). Immunohistochemistry Immunohistochemistry for TIMP3 (ab39184 polyclonal antibody at 1:50 dilution from Abcam, Beverly, MA) was performed in formalin-fixed, paraffin-embedded tissue sections. All slides were stained using the automated Nexes IHC stainer (Ventana, Tucson, AZ) providing consistency of the experimental conditions. Positive and negative controls were run concomitantly in all cases.

J Mol Neurosci (2016) 58:137–144 Table 1 Clinical date of 40 ACTH-secreting pituitary adenoma patients

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Number

Sex

Age (year)

Diameter (mm)

KI-67 (%)

ACTH (pg/ml)

F (ug/dl)

24-h UFC (μg)

1 2

F F

18 34

3 3

1 1

102 63.2

32.45 24.3

632.22 515.31

3

F

45

3

4

66

23.64

462.99

4 5

F M

31 14

3 3

<1 <1

52.1 126

25.16 35.07

201.6 3688.05

6 7

F F

8 30

4 4

1 3

23.6 100

28.54 32.75

175.4 753.6

8

F

42

4

1

36.83

27.3

521.74

9 10

M M

15 37

4 4

2 3

122 149

28.5 42.68

405.84 683.74

11

F

31

4

5

149

39

432.96

12

M

39

4

<1

152

29.84

1015.6

13 14 15 16

F F F F

29 42 48 53

4 4 4 4

1 <1 <1 <1

50.5 36.83 126 57.4

18.18 27.3 26.97 28.59

195.48 521.74 385.84 205

17 18 19

F F F

31 39 27

4 5 5

<1 <1 <1

64.7 65.6 338

42.83 20.54 65.55

764.2 400.2 2512.08

20 21 22 23

F F F F

55 31 30 43

6 6.2 7 7

<1 <1 3 2

61.6 48.7 25.4 45.8

29.51 26.64 29.25 24.58

669.12 243.55 332.28 431.3

24 25 26 27 28

M M M F M

16 23 44 23 48

8 8 8 8 10

1 2 3 5 1

54 69.3 131 102 76.2

30.14 25.01 33.16 30.35 25.73

473.58 519.2 968.4 397.11 152.1

29 30 31

F F F

22 64 65

10 10 10

6 1 <1

80.3 366 271

22 66.31 45.59

116.6 1286.1 1102.08

32 33 34 35 36 37 38 39 40

F F M M F M F F F

31 31 17 36 40 38 34 57 33

10.2 15 17 18 20 23 23 25 50

1 2 3 1 8 1 4 1 1

40.2 48.7 67.8 34.98 48.5 103 63.2 125 116

35.9 52.7 22.28 41.51 22.41 23.23 24.3 28.01 29.79

482.69 243.55 250.38 2006.98 236.8 361.76 515.31 1213.8 342

Statistical Analysis Statistical analysis was performed with SPSS 17.0 software (SPSS Inc, Chicago, IL). TIMP3 mRNA expression was compared between the individual groups by one-way ANOVA. The relationships between TIMP3 mRNA and clinicopathologic characteristics were evaluated by the Mann-Whitney

test. An independent samples t test was used to determine the relationship between invasion and noninvasion. The Spearman rank correlation analysis was used to determine the relationship between TIMP3 mRNA levels and tumor size. And the correlations between TIMP3 mRNA and Ki-67 low index (LI) in ACTH-secreting pituitary adenomas p<.05 (two-tailed) were considered statistically significant.

140 Table 2 Clinical characteristics of the total 50 ACTH-secreting pituitary adenoma patients

J Mol Neurosci (2016) 58:137–144

Characteristics

ANPT group (n=10)

Tumor group (n=40)

P value

Sex, female (no, %) Age (years)

8 (80.0) 34.7 (8.43)

29 (72.5) 34.85 (13.35)

0.824 0.582

Body mass index (>25 kg/m2) no. (%)

6 (60.0)

27 (67.5)

0.898

Hormone level mean (SD) 8AM serum ACTH (pg/ml)

115.68 (84.64)

96.49 (75.92)

0.763

36.85 (13.85)

31.69 (10.81)

0.489

401.22 (307.56) 5.1 (1.29)

670.46 (689.54) 9.31 (9.02)

0.301 0.016

8AM serum free cortisol (ug/dl) 24-h urinary free cortisol (ug/24 h) MRI tumor largest diameter (mm)

Results Expression and Location of TIMP3 in Adenomas and ANPTs

random cases (4 cases for each type). TIMP3 immunoreactivity was weakly localized in the cytoplasma of adenoma cells but strongly in the cytoplasma of ANPTs (Fig. 1). TIMP3 mRNA Expression in Adenomas and in ANPTs

To study the expression and to identify the cellular and subcellular localization of TIMP3 in pituitary adenomas and ANPTs, we performed immunohistochemical staining in 20

Fig. 1 Expression and location of TIMP3 in PAs and ANPTs. a–d Weak immunoreactivity for TIMP3 was detected in the cytoplasm of PAs (a NFA. b GH adenoma. c ACTH adenoma. d PRL adenoma. original magnification ×400). e–f Strongly expressed in the cytoplasm of non-tumorous pituitary tissues cells (original magnification ×200)

TIMP3 mRNA was expressed in all 86 adenoma and 10 ANPT samples (Fig. 2). Using one-way ANOVA, we

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Fig. 2 TIMP3 mRNA levels in the different groups of adenomas and in ANPTs. a TIMP3 mRNA in the ANPT group was significantly different in comparison with each group of the pituitary adenomas (p<.01). TIMP3, tissue inhibitor of metalloproteinase-3, NFA nonfunctional pituitary adenoma, PRL prolactin, ACTH adrenocorticotropic hormone, GH growth hormone, ANPT nontumorous pituitary tissues. b The relevant TIMP3 and GAPDH dissociation curve was appended to show the specific mRNA

found that TIMP3 mRNA expression was significantly different between the different types of PAs and ANPTs (p<0.01 for all comparisons). The levels of expression of TIMP3 were significantly downregulated in the pituitary adenomas compared with those in ANPT.

TIMP3 Protein Expression in ANPTs and in Other Types Pituitary Tumors TIMP3 protein expression was low in most of the 36 nonACTH-secreting pituitary adenoma samples. The results for two ANPTs (lines 1 and 2), 4 GH adenomas, 4 NF adenomas, and 4 PRL adenomas are shown in Fig. 4b.

TIMP3 Protein Expression in ANPTs and in ACTH-Secreting Pituitary Tumors We further measured TIMP3 protein expression by Western blotting to verify the mRNA findings. We employed all of 10 ANPTs as controls and randomly selected 10 pituitary adenomas whose mRNA expression was around the median value of all 40 tumors, to assess TIMP3 protein expression. TIMP3 protein levels were decreased in ACTH-secreting pituitary tumors compared with those in ANPT controls (Fig. 3, p<0.001).

Fig. 3 a TIMP3 protein levels in ACTH-secreting pituitary adenomas and adjacent non-tumorous pituitary tissues assessed by Western blotting. The graph shows the TIMP3 GAPDH relative ratio in 10 tumor samples

TIMP3 mRNA and Protein Expression in Invasive Group and Noninvasive Group of Pituitary Adenomas TIMP3 mRNA levels were 1.92-fold higher in noninvasive PAs than in invasive PAs (Fig. 4a). With the use of an independent samples t test, a statistically significant difference was observed between the two groups (p<0.05). The comparison of TIMP3 protein expression between the different subtypes of noninvasive and invasive pituitary tumors was measured by

and 10 ANPT. p<0.001. b Panel representative Western blots of five tumors and four ANPT samples. ACTH adrenocorticotropic hormone, ANPT non-tumorous pituitary tissue

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Fig. 4 a TIMP3 mRNA expression in invasive and noninvasive PAs. TIMP3 mRNA levels were significantly higher in the noninvasive PAs than in the invasive PAs (p<.05). b TIMP3 protein levels in different subtype pituitary adenomas and adjacent non-tumorous pituitary tissues

assessed by Western blotting, and compared invasive PAs with noninvasive PAs. TIMP3 tissue inhibitor of metalloproteinase-3, NF nonfunctional adenoma, PRL prolactin, GH growth hormone, ANPT non-tumorous pituitary tissue

Western blotting (Fig. 4b). The expression trends of TIMP3 protein were stronger in noninvasive pituitary adenomas compared with invasive pituitary adenomas (Fig. 4b).

(Fig. 5). With the use of an independent sample t test, a statistically significant difference was observed between the two groups (p=0.0265).

TIMP3 mRNA in Microadenomas, Macroadenomas, and Giant Adenomas

Association Between TIMP3 mRNA and the Clinicopathologic Characteristics of ACTH-Secreting Pituitary Adenomas

TIMP3 mRNA expression was markedly lower with larger tumors. Although there were not statistically significant difference between macroadenomas and microadenomas or giant adenomas, TIMP3 mRNA levels were 2.58-fold higher in microadenomas than in giant adenoma group Fig. 5 TIMP3 mRNA levels in microadenoma group, macroadenoma group, and giant adenoma group of PAs TIMP3 mRNA in the microadenomas group was significantly different in comparison with giant adenomas group (p<.05). b The relevant TIMP3 and GAPDH dissociation curve was appended to show the specific mRNA

The associations between TIMP3 mRNA and the clinicopathologic characteristics of ACTH-secreting pituitary adenomas are described in Table 2. Using the unpaired t test, there was no significant difference in TIMP3 mRNA expression

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between sex, age, BMI, or hormone secretion. Specifically, there was no correlation between TIMP3 mRNA and serum ACTH, cortisol, or 24-h urine free cortisol levels in ACTHsecreting pituitary tumors. TIMP3 mRNA expression was inversely correlated with tumor maximum diameter (Rho= −0.378; p=0.016). The Spearman correlation coefficient for TIMP3 mRNA and Ki-67 LI was −0.322 (p<.05), indicating that TIMP3 mRNA expression is negatively correlated with Ki-67 labeling index of ACTH-secreting pituitary tumors.

how TIMP3 downregulation plays a role in promoting growth and invasiveness of pituitary adenomas. In conclusion, TIMP3 was significantly under-expressed in pituitary adenomas. In pituitary adenomas, lower TIMP3 levels were significantly correlated with tumor size, Ki-67 LI, and invasiveness. Therefore, TIMP3 may play an important role in the development and progression of invasive PAs, and may be a valuable molecular biomarker for the diagnosis of pituitary adenomas and the evaluation of their level of invasiveness.

Discussion

Acknowledgments The authors thank Dr. Run Yu (Cedars-Sinai Medical Center, Los Angeles, CA) and Shunzeng Lv for the helpful discussion and editing of this manuscript.

The matrix metalloproteinases are a family of zinc proteases involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, tissue and bone remodeling, wound healing, and angiogenesis (Shen et al. 2010; Zerrouqi et al. 2012; Nieuwesteeg et al. 2012; Fox et al. 2014). Within the extracellular matrix, the tissue inhibitors of metalloproteinases (TIMP1–4) inhibit the activity of matrix metalloproteinases (Beaulieu et al. 1999). The human tissue inhibitor of metalloproteinase-3 (TIMP3) gene, a natural inhibitor for matrix metalloproteinase (MMP), has complicated roles in tumorigenesis but has not been studied in pituitary adenomas. In this study, we addressed TIMP3 expression and the clinical significance of TIMP3 expression in a large number of pituitary adenomas. As congruent results were obtained with multiple methods of determining TIMP3 expression including immunohistochemistry, Western blot, and RT-PCR, our results likely represent true TIMP3 expression in pituitary adenomas. We found that TIMP3 was under-expressed in pituitary adenomas, compared to ANPTs. To the best of our knowledge, this is the first study to confirm the differential expression of TIMP3 in all types of pituitary adenomas. Our data indicate that TIMP3 protein is predominantly expressed in the cytoplasm of pituitary adenoma cells and normal pituitary gland cells. As TIMP3 expression was even lower in invasive pituitary adenomas and TIMP3 expression was negatively correlated with Ki-67 labeling index and tumor size, shown in this study, TIMP3 may have a role in the tumorigenesis and progression of pituitary adenomas. Decreased TIMP3 expression has been observed in a variety of cancers such as kidney, gastric, colon, and endometrioid endometrial carcinomas. Various studies propose that the reduced expression of TIMP3 contributes to the metastatic process, and other studies suggest that TIMP3 overexpression reduces metastasis, induces apoptosis, and augments drug sensitivity. The mechanisms in which TIMPs exert a tumor suppressor function are reported. Our results in this study support a similar tumor suppressor function of TIMP3 in pituitary adenomas, and further studies are needed to address how TIMP3 levels are downregulated in pituitary adenomas and

Compliance with Ethical Standards Conflict of Interest The authors declare have no competing interests. Ethics Approval The study was approved by the Ethical Committee of Peking union medical college hospital, and all patients had written informed consent.

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