G Protein γ7 Expression as a New Clinicopathological Marker in Patients With Intrahepatic Cholangiocarcinoma

Hypothesis  The signal alterations mediated by small G proteins such as Ras, Rho, and Rac have been reported in several cancers. The human G protein γ7 (G-γ7) gene, which is down-regulated in various digestive organ cancers, was recently identified and cloned. Thus, the G-γ7–coupled heterotrimeric G proteins may also contribute to carcinogenesis in human cancers.

Setting  University hospital and medical institute of bioregulation.

Patients and Methods  The clinicopathological significance of G-γ7 expression in 18 patients with intrahepatic cholangiocarcinoma (IHCC) was examined. The tumor-nontumor ratio of G-γ7 expression was determined using reverse transcription polymerase chain reaction analysis. To visualize the localization of G-γ7, an immunohistochemical study was performed.

Main Outcome Measure  Clinicopathological significance of G-γ7 expression in human IHCC.

Results  Expression of G-γ7 messenger RNA was lower in tumor tissue than in the corresponding nontumor tissue in 17 (94%) of 18 patients with IHCC. The mean tumor-nontumor ratio was 0.54. Eleven patients with tumor-nontumor ratios less than 0.5 showed significantly poorer differentiated IHCC than 7 with tumor-nontumor ratios of 0.5 and greater (P<.01). Decreased expression of G-γ7 protein in the carcinoma tissue, especially in the poorly differentiated IHCC tissue, was confirmed using immunohistochemical staining.

Conclusions  Reduced expression of G-γ7 is associated with the histological grade of IHCC and may therefore prove to be a useful marker for predicting the biological aggressiveness of human IHCC.

THE TECHNIQUES of subtractive complementary DNA cloning and differential display between tumor and normal tissue samples of gastrointestinal tract cancers have been used to determine new clinicopathological and prognostic molecular markers.^1-3 Accordingly, Shibata et al⁴ recently identified and cloned the human G protein γ7 (G-γ7) gene, which is down-regulated in pancreatic cancer and cell lines. Shibata et al⁵ further confirmed a decrease in the expression of G-γ7 in other gastrointestinal tract cancers, including esophageal, gastric, and colorectal cancers.

Heterotrimeric G protein, which is composed of α, β, and γ subunits, transduces the signals across the plasma membrane from a receptor to an effector.^6-8 The signal-transducing elements of the G protein are not only the α subunit that binds and hydrolyzes guanosine 5′-triphosphate, but also the β γ subunit, which plays a major role in signal transmission.^9,10 To date, 7 β subunits and 11 γ subunits have been identified in mammalian systems.¹¹ The G protein γ subunits determine the functional specificity and stabilize the heterotrimeric G protein to the cellular membrane.^6,12,13 Although the signal alterations mediated by small G proteins such as Ras,¹⁴ Rho,^15,16 and Rac¹⁵ have been reported in several cancers, little information is available on the changes in the mediated signals of heterotrimeric G proteins in cancer. Because the G protein β γ subunit clearly controls the signals involved in cell growth¹⁷ and the G-γ7 gene is widely distributed in the signal transduction pathways,^18,19 the G-γ7–coupled G proteins may contribute to carcinogenesis in many kinds of cancers.

In a previous study, Shibata et al⁵ transfected the G-γ7 complementary DNA into a human esophageal carcinoma cell line to determine its biological role in cancer. Expression of G-γ7 suppressed cell growth when the cells were confluent. Moreover, G-γ7 expression suppressed tumorigenicity in nude mice, thus implicating the biological effects of G-γ7 in vivo. All these findings prompted us to analyze the clinical significance of this gene in human cancer. In the present study, we determined G-γ7 expression and its clinical significance in patients with intrahepatic cholangiocarcinoma (IHCC) who underwent hepatic resection. This study showed a significant association between G-γ7 expression and the histological grade of IHCC, which is one of the most important prognostic factors for patients with IHCC.^20,21

Eighteen patients with IHCC who underwent surgery at the Medical Institute of Bioregulation Hospital and the Department of Surgery and Science, Kyushu University, were studied. All 18 patients underwent hepatectomies for primary tumors. No patients demonstrated complications with either hepatolithiasis or primary sclerosing cholangitis. After the hepatectomies, the tumor and corresponding nontumor tissues were immediately frozen in liquid nitrogen and kept at −90°C until use. Written informed consent was obtained from all patients. The study was performed according to the latest revision of the Helsinki Declaration (1989) for human research. The human bile duct cancer cell lines, HuCC-T1, TFK-1, and HuH28, were provided by the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Tohoku, Japan.

Total RNA was prepared using a modification of the guanidinium thiocyanate method described elsewhere.^1,2 To avoid contamination by genomic DNA, 50 µg of total RNA was treated with 1 U of deoxyribonuclease I (Message Clean Kit; Gene Hunter, Nashville, Tenn). The treated RNAs were dissolved to 1 µg/µL using diethylpyrocarbonate-treated water and then were stored at −90°C until use.

Complementary DNA was synthesized from 2.5 µg of total RNA.^4,5,22 The oligonucleotide primer pairs for G-γ7 were synthesized (sense primer: 5′-GTCAACGGATTTGGTCTGTAT-3′; antisense primer: 5′-AGTCTTCTGGGTGGCAGTGAT-3′). Polymerase chain reaction (PCR) was performed using a method described previously.^4,5,22 To ensure that the RNA was of sufficient purity for reverse transcription (RT)-PCR, a PCR assay with primers specific for the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complementary DNA was carried out in each case. Aliquots of the PCR-amplified DNA were electrophoresed on 1.5% agarose gels containing ethidium bromide. Expression of G-γ7 and GAPDH was evaluated by computer-assisted image analysis. The tumor-nontumor ratio (T/N ratio) of G-γ7 expression was calculated after correcting for the T/N ratio of GAPDH expression. Values of 0.5 or greater were considered positive, and values less than 0.5 were considered negative.

All samples for immunohistochemical staining were fixed in buffered formalin, embedded in paraffin, and cut into 5-µm-thick slices. The G-γ7 protein was detected using antibovine G-γ7 (Santa Cruz Biotechnology Inc, Santa Cruz, Calif) followed by the streptavidin-biotin-peroxidase method (LSAB Kit; DAKO Japan Co, Ltd, Kyoto, Japan), as described previously.^4,5,23

All the clinical data variables were available for evaluation. The clinicopathological features were evaluated according to the General Rules for the Clinical and Pathological Study of Primary Liver Cancer.²⁴ The data were then compared between G-γ7–positive and G-γ7–negative patients.

For continuous variables, data are expressed as mean ± SD. To compare the clinicopathological characteristics between the 2 patient groups, either the unpaired t test or the χ² test was used. A 2-sided P<.05 was considered significant.

The T/N ratios of G-γ7 messenger RNA (mRNA), which were corrected for those of GAPDH mRNA, ranged from 0.20 to 1.69 (mean, 0.54) and were larger than 1.0 in only 1 patient (6%) (Table 1). Consistent with previous findings in other gastrointestinal tract cancers,⁵ expression of G-γ7 mRNA in IHCC was markedly lower in tumor tissues than in the corresponding nontumor tissues. Figure 1 shows representative patients. All 3 cell lines (HuCC-T1, TFK-1, and HuH28) showed no expression of G-γ7 mRNA by RT-PCR (Figure 2), which is also consistent with findings from a previous study⁴ of the pancreatic cancer cell lines.

Staining of G-γ7 was markedly weaker in IHCC tissue than in the corresponding nontumor hepatic tissue in moderately differentiated and poorly differentiated IHCC (Figure 3). Almost completely negative staining in poorly differentiated IHCC tissue was observed (Figure 3B). The immunohistochemical results closely corresponded with the RT-PCR results.

The clinicopathological features analyzed in relation to the G-γ7 expression status are given in Table 2. The RT-PCR results indicated that 11 patients (61%) were negative and 7 (39%) were positive. There were no significant differences between G-γ7 expression and host factors, such as age, sex, and preoperative liver function test results. In contrast, G-γ7 expression strongly correlated with histological grade. In poorly differentiated IHCC tissues, G-γ7 expression was significantly lower than that in moderately differentiated IHCC tissues (P<.01). The histological grade of IHCC and the T/N ratio of G-γ7 expression in individual patients are listed in Table 1. However, other pathological variables, such as tumor diameter, intrahepatic metastasis, and lymph node metastasis, were not associated with the G-γ7 expression status.

Identification of the genes that are specifically expressed in either tumor or nontumor tissue is considered to be important for understanding the biological mechanisms of cancer. At our institution (Medical Institute of Bioregulation, Kyushu University), Shibata et al⁵ recently identified and cloned the human G-γ7 gene, which is generally down-regulated in digestive organ cancers. In the present study, we thus studied the clinical significance of such G-γ7 expression in one type of human cancer: IHCC.

The signal alterations mediated by small G proteins in various cancers are already well known.^14-16 Regarding heterotrimeric G protein, the oncogenic mutations of G protein α subunit genes have also been documented in some endocrine tumors, including pituitary,²⁵ adrenal,²⁶ and thyroid²⁷ tumors. However, to the best of our knowledge, there is no information on the oncogenic mutations of the G protein β γ subunits in human cancers. Shibata et al⁵ previously found that a stable transfection of a carcinoma cell line with a G-γ7 expression vector inhibited cell growth and tritiated thymidine uptake when the cells were 100% confluent. Furthermore, the G-γ7–induced G0/G1 arrest at high cell densities was associated with p27^Kip1 expression. Our findings suggested that G-γ7–coupled heterotrimeric G proteins might transduce a growth inhibition signal with cell contact in normal cells, but this does not occur in cancer because of the inactivation of G-γ7.⁵ In the present study, we confirmed that the expression of G-γ7 mRNA was markedly suppressed in human IHCC tissue as well. This decreased gene expression significantly correlated with the histological grade of IHCC (Table 2). The results of immunohistochemical staining also indicated that G-γ7 expression in poorly differentiated IHCC was weaker than that in moderately differentiated IHCC. Although we are aware that only a few cases have been investigated, this is the first study, to our knowledge, to suggest that the signal alterations mediated by the β γ subunits of heterotrimeric G protein may affect the clinical features of human cancer.

The precise mechanisms by which the changes in the signaling pathway of G-γ7–coupled heterotrimeric G proteins were associated with the histological grade of IHCC remain to be established. However, according to the results of immunohistochemical analysis, the poorly differentiated IHCC with a solid pattern had an apparently higher cell density than the moderately differentiated IHCC with a granular pattern (Figure 3). It is possible that the G-γ7 signaling in poorly differentiated IHCC is inactivated even more than that in moderately differentiated IHCC because of the higher cell density. Alternatively, these tumor cells with a solid formation might be able to escape cell-cell contact-induced growth inhibition and even grow rapidly in the absence of or with a small presence of G-γ7 transduction signaling. On the other hand, Durand et al²⁸ demonstrated that p27^Kip1 protein level is high when the cells stop dividing and terminally differentiate during oligodendrocyte development, thus suggesting a strong correlation between cell differentiation and the accumulation of p27^Kip1.²⁹ Neural differentiation has also been reported to be responsible for the on-off switch of the expression of G protein γ subunits.³⁰ Taken together, these findings, along with findings from a previous study of p27^Kip1 expression in the G-γ7 transfectants,⁵ the markedly reduced signaling in the G-γ7 transduction pathway might also be associated with an absence of p27^Kip1-induced growth arrest.

In conclusion, expression of G-γ7 mRNA was down-regulated in IHCC tissue and was clinically associated with the poorer histological grade of IHCC. Results of an immunohistochemical study also showed a drastically suppressed degree of staining in poorly differentiated IHCC tissue. The histological grade of IHCC is one of the most important prognostic factors for patients with IHCC.^20,21 Therefore, the G-γ7 expression status may be a significant indicator for identifying the malignant biological behavior of human IHCC. However, the precise function of G-γ7 in IHCC tissue remains unclear. Further investigation is needed to clarify the relationship between G-γ7 and carcinoma progression.

We thank Kohei Shibata, MD, for helpful discussion and Kenji Sato, MS, Toshiko Shimooka, MS, Junko Miyake, MS, and Kazue Ogata, MS, for their excellent technical assistance.

Corresponding author and reprints: Masaki Mori, MD, Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu 874-0838, Japan (e-mail: mmori@tsurumi.beppu.kyushu-u.ac.jp).