Dual function of transforming growth factor beta 2 in the progression of gastric carcinoma

Abstract

BACKGROUND

It has been reported that transforming growth factor beta (TGFβ) might play an important role for the development of various types of carcinomas. TGFβ is produced from not only cancer cells but also stromal cells such as fibroblasts. Controversial functions of TGFβ for tumor progression such as tumor suppression and progression has been widely recognized. TGFβ has 3 subtypes including TGFβ1, TGFβ2, and TGFβ3. The role of TGFβ1 and TGFβ3 for gastric carcinoma (GC) progression has been well-known, but that of TGFβ2 remains to be unknown.

AIM

To clarify the significance of TGFβ2 in the development of GC.

METHODS

Immunohistochemical analysis of GC was performed to clarify the clinical function of TGFβ2 on the progression of GC by evaluating TGFβ2 expression on both cancer cells and cancer-associated fibroblasts (CAFs) using 518 GC specimens.

RESULTS

High TGFβ2 expression in cancer cells was significantly correlated with T factor and stage, but not with vascular invasion and lymphatic invasion. In contrast, TGFβ2 expression on CAFs was significantly correlated with lymph node metastasis and vascular invasion, but not with the T factor. Five-year survival of patients with TGFβ2-high on cancer cells and -low on CAFs was significantly worse in compared to the other group These findings suggest that TGFβ2 might play differential roles between cancer cells and CAFs in the development of GC.

CONCLUSION

TGFβ2 on cancer cells might stimulates the cancer proliferation and invasion, but not TGFβ2 on CAFs. TGFβ2 high on cancer cells and low on CAF or others might be a promising prognostic factor for patients with GC.

Key Words: Transforming growth factor beta 2; Gastric cancer; Cancer-associated fibroblasts; Tumor microenvironment

Core Tip: The immunohistochemical analysis of gastric carcinoma (GC) was performed to clarify the clinical function of transforming growth factor beta 2 (TGFβ2) on the progression of GC. TGFβ2 expression on cancer-associated fibroblasts (CAFs) was significantly correlated with lymph node metastasis and vascular invasion, but not with the T factor. Five-year survival of patients with TGFβ2-high on cancer cells and -low on CAFs was significantly worse in compared to the other group TGFβ2 on cancer cells might stimulates the cancer proliferation and invasion, but not TGFβ2 on CAFs. TGFβ2 high on cancer cells and low on CAF or others might be a promising prognostic factor for patients with GC.

INTRODUCTION

Despite of the current development of therapeutic tools for carcinomas, gastric carcinoma (GC) remains to be one of leading causes of cancer death[1]. The mechanism of metastasis of GC has not been clarified, so far, while the main cause of cancer-related death has been distant metastasis[2,3].

It has been reported that transforming growth factor beta (TGFβ) is closely associated with the cancer cell proliferation and differentiation[4]. TGFβ family consists of TGFβ1, TGFβ2 and TGFβ3[4]. TGFβ subclasses have high amino acid sequence conservation, and shows several differences among three subtypes in expression profiles, function domain activation patterns, and activation threshold[5-7]. Each TGFβ subtype shows characteristic biologic function for tumor development[5-8]. TGFβ family has been suggested to be a promising therapeutic target for GC[9,10], however the useful drug for TGFβ subtype has not developed, so far. It is necessary to further understanding of role of characteristic function of TGFβ subtypes on the distant metastasis of GC.

Cancer-associated fibroblasts (CAFs) have been reported to be one of important stromal cells in cancer progression[11-13]. Role of TGFβ2 in the development of cancer remains to be controversial as tumor promotion or tumor inhibition[14-16], however, there is no report which clarified the significance of TGFβ2 expression on cancer cell and CAFs. Then, this study aimed to clarify the clinicopathologic significance of TGFβ2 in the development of GC, in view to TGFβ2 expression on cancer cells and on CAFs.

MATERIALS AND METHODS

A total of 518 GC specimens, those were surgically resected from GC patients at Osaka Metropolitan University Hospital, was collected from 2010 to 2018. In 518 GC cases, 358 were men and 160 were women. None of the patients had undergone preoperative radiotherapy. The pathologic diagnoses and classifications were made according to the Union for International Cancer Control tumor-node-metastasis classification of malignant tumors[17]. All tumors were staged in according to tumor-node-metastasis classification of malignant tumors and graded according to a modification in grading system proposed by World Health Organization for gastric tumors[17]. Patents were followed for a period ranging from 1 month to 60 months. Recurrence-free survival (RFS) was analyzed for patients with stage I-III disease and was calculated from baseline to recurrence, metastasis, or death from any cause. Progression-free survival was analyzed for patients with stage IV disease and for those with stage I-III disease from the time of first recurrence or metastasis. Tumor response was defined as the interval from the first confirmed recurrent metastasis to disease progression or death from any cause. Tumor response was evaluated according to the Response Evaluation Criteria in Solid Tumors version 1.1[18,19]. This study was approved by the Osaka Metropolitan University Ethics Committee, approval No. 2022-111, No. 924, and No. 2022-077. Informed consent was obtained in writing from all patients, and the study was conducted according to the principles of the Declaration of Helsinki.

Immunohistochemistry

The expression of TGFβ2 was evaluated by immunohistochemistry using 4 μm thick tissue. They were dewaxed in xylene and hydrated through a series of graded concentrations of alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes. For antigen retrieval, the slide sections were autoclaved at 105 degrees Celsius for 10 minutes. Subsequently, a standard streptavidin-biotin-peroxidase technique (Nichirei, Tokyo, Japan) was applied to detect the antigens. Mouse polyclonal antibodies to TGFβ2 were used at 1:400 dilution (Santa Cruz Biotechnology Inc. Louis, MO, United States). Sections were counterstained with Harris hematoxylin. All procedures were performed at room temperature.

Morphometric analysis

TGFβ2 expression was evaluated separately at cancer cells and at CAFs in GC tissue. Each TGFβ2 expression levels were evaluated by the staining intensity and the ratio of stained cells. The intensity of TGFβ2 staining was scored as 4-point scales from 0 to 3 (0, no one; 1, weak; 2, moderate; 3, strong). The range of positive staining was assessed as a percentage (0%-100%) and classified into 4 classes as scored 0-3 (0, 0%; 1, 1%-30%; 2, 31%-70%; 3, 71%-100%). The combination of intensity and range was used to assign slides to the low or high expression group. Both intensity and range were evaluated within each cellular region of cancer cells and CAFs. All slides were screened and scored using a blinded method without knowledge of clinical pathological data.

Statistical analysis

All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). Intergroup comparisons regarding correlation of pathologic data with stanning results were performed using Mann-Whitney U test. The Kaplan-Meier procedure was also used to compare the survival curves. A P value < 0.05 was considered significant.

The multivariate analysis was performed by first analyzing each variable in the univariate analysis and then validating all variables with P value < 0.05 before performing the multivariate analysis. Therefore, the P values were calculated using EZR and the Cox proportional hazards model.

RESULTS

TGFβ2 expression in GC

During the follow-up period after the surgery, 356 (68.7%) of all 518 patients were alive. Of 518 patients, 202 cases were classified as stage I, 122 cases were stage II, 141 cases were stage III, 53 cases were stage IV. Two-hundred forty-three cases were histologically assigned as well-differentiated, and 240 cases were poor-differentiated or signet cell. TGFβ2 expression pattern in GC tissue was immunohistochemically shown in Figure 1. TGFβ2 expression was observed at the cell cytoplasm in cancer cells and CAFs.

Figure 1 Representative transforming growth factor beta 2 expression in gastric cancer. Transforming growth factor beta 2 (TGFβ2) was expressed at the cytoplasm of cancer cells and cancer-associated fibroblasts (CAFs). A: Both cancer cells and CAFs expressed TGFβ2; B: CAFs expressed TGFβ2, but not cancer cells; C: CAFs expressed TGFβ2, but not cancer cells; D: TGFβ2 was not expressed on cancer cells and CAFs. (Bar: 100 μm. Black arrows: TGFβ2 positive cancer cells; blue arrows: TGFβ2 positive CAFs) TGFβ2: Transforming growth factor beta 2; HE: Hematoxylin and eosin.

TGFβ2 expression on cancer cells

Table 1 shows the clinicopathological characteristics of the patients and TGFβ2 expression on cancer cells and CAF in GC. TGFβ2 expression on cancer cell was divided into two groups as a low-expression group (score: 0-2, n = 317) and a high-expression group (score: 3-9, n = 201) in according to TGFβ2 expression score (0-9). The high-expression group had significantly higher pT stage compared to the low-expression group (P = 0.0193: T1, T2, T3 vs T4). No significant difference was observed in pN, metastasis (M), lymph node metastasis or venous invasion of cancer.

Table 1 Transforming growth factor beta 2 expression in relation to various pathologic parameters.

Parameters	Cancer cell			CAF			Cancer cell and CAF
	Low (n = 317)	High (n = 201)	P value	Low (n = 238)	High (n = 280)	P value	High on cancer cell and low on CAF (n = 70)	The others (n = 448)	P value
Gender
Male (n = 358)	218	140	-	154	204	-	43	315	-
Female (n = 160)	99	61	0.909	84	76	0.0567	27	133	0.175
Pathology
Tubular adenocarcinoma (n = 243)	137	106	-	113	130	-	38	205	-
Poorly differentiated or signet-ring cell (n = 240)	157	83	0.105	110	130	0.72	28	212	0.38
TMN
T1 (n = 161)	114	47	-	67	94	-	16	145	-
T2 (n = 87)	51	36	-	36	51	-	12	75	-
T3 (n = 126)	76	50	-	67	59	-	21	105	-
T4 (n = 144)	76	68	-	68	76	-	21	123	-
T1, T2, T3 (n = 374)	241	133	-	170	204	-	49	325	-
T4 (n = 144)	76	68	0.0193	68	76	0.792	21	123	0.765
N0 (n = 248)	160	88	-	98	150	-	27	221	-
N1 (n = 91)	52	39	-	50	41	-	18	73	-
N2 (n = 72)	41	31	-	32	40	-	9	63	-
N3 (n = 104)	62	42	-	56	48	-	16	88	-
N0 (n = 248)	160	88	-	98	150	-	27	221	-
N1, N2, N3 (n = 267)	155	112	0.317	138	129	0.0167	43	224	0.177
M0 (n = 465)	286	179	-	206	259	-	61	404	-
M1 (n = 53)	31	22	0.781	32	21	0.0376	9	44	0.57
Pathological findings of the primary tumor
Infiltration
A (n = 26)	18	8	-	20	6	-	6	20	-
B (n = 354)	212	142	-	154	200	-	48	306	-
C (n = 115)	72	43	-	56	59	-	14	101	-
A, b (n = 380)	230	150	-	174	206	-	54	326	-
C (n = 115)	72	43	0.85	56	59	0.47	14	101	0.673
Lymphatic invasion
0 (n = 201)	136	65	-	87	114	-	25	176	-
1 (n = 153)	93	60	-	68	85	-	19	134	-
2 (n = 108)	56	52	-	55	53	-	18	90	-
3 (n = 54)	31	23	-	27	27	-	8	46	-
0 (n = 201)	136	65	-	87	114	-	25	176	-
1, 2, 3 (n = 315)	160	135	0.0544	150	165	0.624	45	270	0.715
Vanous invasion
0 (n = 365)	228	137	-	152	213	-	49	326	-
1 (n = 110)	65	45	-	60	50	-	14	90	-
2 (n = 33)	17	16	-	21	12	-	7	26	-
3 (n = 6)	5	1	-	3	3	-	0	6	-
0 (n = 365)	234	141	-	159	216	-	49	326	-
1, 2, 3 (n = 149)	81	58	0.625	77	62	0.0314	21	118	0.609

For all parameters, the classification into low/high was based on transforming growth factor beta 2 expression observed in immunohistochemical staining of the primary tumor. The P values for each parameter were the P values between the two groups, the low and high groups, for the cancer cell and cancer-associated fibroblast groups, respectively. In some parameters, some missing data of transforming growth factor beta 2 expression were deleted from the description. CAF: Cancer-associated fibroblast; TNM: Tumor-node-metastasis.

TGFβ2 expression on CAFs

TGFβ2 expression on CAFs was also divided into two groups as a low-expression group (score: 0-1, n = 238) and a high-expression group (score: 2-9, n = 280). TGFβ2-high expression group was significantly associated with smaller pN stage, in compared to TGFβ2-low-expression group (P = 0.0167: PN0 or pN1-3). Metastasis was significantly more frequent in the low-expression group (P = 0.0376: M0 or M1). The high-expression group had less venous invasion compared to the low-expression group (P = 0.0314: V0 or v1-3) (Table 1).

Correlation between TGFβ2 expression and the patients’ survival

TGFβ2 expression in the GC was classified into 2 patterns, as follows. One is the combination of high-TGFβ2 at cancer cell and low-TGFβ2 in CAF, the other is the combination of high-TGFβ2 in cancer cell and low-TGFβ2 in CAF. Five-year overall survival (OS), and RFS were significantly different (OS: P = 0.0382, RFS: P = 0.0206) between the two groups (Figure 2). OS curve indicated that the two groups followed same curves during the first 2 years. In contrast, the survival rate of high-TGFβ2 in cancer cell and low-TGFβ2 in CAF group showed significantly worse.

Figure 2 Survival. Survival rate of patients with gastric cancer was evaluated between the transforming growth factor beta 2 (TGFβ2)-high on cancer cells and -low on cancer-associated fibroblast (CAF) group, and the others group. Solid lines represent TGFβ2-positive on cancer cells and TGFβ2-negative on CAF, and dotted lines represent TGFβ2-negative on cancer cells or TGFβ2-positive on CAF. A: Both overall survival and recurrence-free survival curves showed a worse prognosis in the combination group after one year. Five-year overall survival indicated that TGFβ2-high on cancer cells and -low on CAF group showed significantly poor prognosis in compared to the other group (P = 0.0382); B: Recurrence-free survival showed that TGFβ2-high on cancer cells and -low on CAF group had a significant worse prognosis in compared to the other group (P = 0.0206). TGFβ2: Transforming growth factor beta 2; CAF: Cancer-associated fibroblast.

Univariate and multivariate analysis

Univariate analysis for OS and RFS was performed on the factors including pT, pN, M and the combination of high-TGFβ2 on cancer cell and low-TGFβ2 on CAF, that showed significant difference in clinicopathological background, to determine 5-year OS and RFS. Multivariate analysis for OS and RFS was performed to factors that showed significant difference by Univariate analysis. The high-TGFβ2 on cancer cell and low-TGFβ2 on CAF was an independent factor in RFS (Table 2).

Table 2 Univariate and multivariate statistics with survival (Cox proportional hazard regression analysis model).

Variables		Univariate	Multivariate
		P value	HR (95%CI)	P value
OS	Gender	0.9314	-	-
	His (tub: 0, por/sig: 1)	0.1137	-	-
	pT (pT1-3 vs pT4)	< 0.001	2.201 (1.5260-3.175)	< 0.001
	pN (pN0 vs pN1-3)	< 0.001	1.412 (0.9173-2.173)	0.117
	M (M0 vs M1)	< 0.001	3.140 (2.0720-4.758)	< 0.001
	inf (inf a-b vs inf c)	0.1699	-	-
	ly (ly0 vs ly1-3)	< 0.001	1.807 (1.1400-2.865)	0.01187
	v (v0 vs v1-3)	< 0.001	1.235 (0.8764-1.739)	0.2281
	TGFβ2 expression on cancer cell	0.1964	-	-
	TGFβ2 expression on CAF	0.5104	-	-
	TGFβ2 expression high on cancer cell and low on CAF or the others	0.03974	1.448 (0.9596-2.186)	0.07776
RFS	Gender	0.7913	-	-
	His (tub: 0, por/sig: 1)	0.5772	-	-
	pT (pT1-3 vs pT4)	< 0.001	2.457 (1.733-3.482)	< 0.001
	pN (pN0 vs pN1-3)	< 0.001	1.682 (1.124-2.516)	0.0114
	inf (inf a-b vs inf c)	0.1504	-	-
	ly (ly0 vs ly1-3)	< 0.001	1.613 (1.054-2.468)	0.0276
	v (v0 vs v1-3)	< 0.001	1.467 (1.037-2.074)	0.03025
	TGFβ2 expression on cancer cell	0.1639	-	-
	TGFβ2 expression on CAF	0.7394	-	-
	TGFβ2 expression high on cancer cell and low on CAF or the others	0.02194	1.637 (1.077-2.489)	0.02113

Univariate analyses were performed on overall survival and recurrence-free survival in 2-presents statistics for each parameter. Multivariate analyses were then performed on all variables that showed significant differences in the univariate analyses, and their independence factors were examined using the Cox proportional hazards model. CI: Confidence interval; HR: Hazard ratio; OS: Overall survival; TGFβ2: Transforming growth factor beta 2; CAF: Cancer-associated fibroblast; RFS: Recurrence-free survival; inf: Infiltration; ly: Lymphatic invasion; v: Vanous invasion.

DISCUSSION

Accumulated research suggests that TGFβ functions differently depending on the cell type and context. Generally, TGFβ regulates cell survival, metabolism, growth, proliferation, differentiation, adhesion, migration, and cell death[20].

On the one hand, it has been reported that TGFβ2 exists in the chief cells of gastric epithelial cells, and in GC cells[16,21,22]. In this study, TGFβ2 staining was observed not only in GC cells but also in CAF, which indicated that CAF might produce TGFβ2. These findings suggest that TGFβ2 from GC cells but also from CAF might promote tumor invasion and metastasis at advanced stage[14]. The significant difference of TGFβ2 expression on GC cells might indicated that TGFβ2 plays an important role for GC progression.

On the other hand, TGFβ2 expression on CAF was significantly associated with lower pN stage, which might suggest that TGFβ2 secretion from CAF is promoted in advanced tumor stage, and TGFβ2 from CAF stimulates the lymphatic and vascular invasion by cancer cells. GC cells and CAFs expressed TGFβ2 at lower stage. TGFβ2 produced from CAF might be transferred cancer cell as the tumor progression. The TGFβ2 transfer might be associated to cancer invasion. TGFβ2 might show different role between cancer cells and CAFs. The localization of TGFβ2 may different and diversity between the 2 groups, as the time course of tumor progression. TGFβ2 in cancer cell and CAF differences respectively in cancer progression and factors involved in metastasis. TGFβ2 might play differential roles between cancer cells and CAFs in the development of GC.

Survival rate of patients was evaluated between the TGFβ2-high on cancer cells and -low on CAF group, and the others group. The difference of survival was significantly evident after two years. TGFβ2 may have a slow lymphatic and vascular invasion promoting effect that is related to recurrence and metastasis two years or more after the first operation.

As limitation of this study, no in vivo experiment was performed to confirm the significance of TGFβ2 in the development of GC. We would plan to investigate the differences in the roles of TGFβ2 in cancer cells and CAFs in vivo studies using animal models in the future.

CONCLUSION

TGFβ2 might play differential roles between cancer cells and CAFs in the development of GC. TGFβ2 on cancer cells might stimulates the cancer proliferation and invasion, but not TGFβ2 on CAFs. TGFβ2 high on cancer cells and low on CAF or others might be a promising prognostic factor for patients with GC.