Ann Surg Oncol DOI 10.1245/s10434-015-4485-4

ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS

Expressional STAT3/STAT5 Ratio is an Independent Prognostic Marker in Colon Carcinoma Fee Klupp, MD1, Johannes Diers, MD1, Christoph Kahlert, MD1, Lena Neumann, MD1, Niels Halama, MD2, Clemens Franz, MD1, Thomas Schmidt, MD, PhD1, Felix Lasitschka, MD4,5, Arne Warth, MD4, Juergen Weitz, MD3, Moritz Koch, MD3, Martin Schneider, MD1, and Alexis Ulrich, MD1 Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany; 2National Center for Tumor Diseases, Medical Oncology, and Internal Medicine VI, Tissue Imaging and Analysis Center, Bioquant, University of Heidelberg, Heidelberg, Germany; 3Department of Visceral, Thoracic, and Vascular Surgery, University of Dresden, Dresden, Germany; 4Institute of Pathology, University of Heidelberg, Heidelberg, Germany; 5Tissue Bank of the National Center for Tumor Diseases (NCT), Heidelberg, Germany 1

ABSTRACT Background. Signal transducer and activator of transcription proteins (STATs) are crucial regulators of cell growth and differentiation; however, their specific prognostic impact in human colon cancer has only been studied to limited extent. We aimed to assess the prognostic significance of specific STAT expression patterns in colon carcinoma. Methods. Protein expression patterns of activated STAT1, STAT3, STAT4, and STAT5 in human colon carcinoma tissue and corresponding healthy mucosa (n = 104) were assessed using multiplex bead-based immunoassay technologies. Expression patterns were correlated with clinical and survival data. Immunohistochemistry was performed to assess spatial expression of STAT3 and STAT5. Results. STAT3 was underexpressed whereas STAT4 and STAT5 were overexpressed in colon carcinoma tissue. Primary tumors from patients with distant metastases (M1) displayed significantly increased expression of STAT1 and STAT3 but decreased expression of STAT4 and STAT5. Increased tumor expression of STAT1 or STAT3 was associated with impaired patient survival, whereas increased

Electronic supplementary material The online version of this article (doi:10.1245/s10434-015-4485-4) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2015 First Received: 5 August 2014 M. Schneider, MD e-mail: [email protected]

expression of STAT4 or STAT5 correlated with improved survival. Multivariate analysis identified an increased STAT3/STAT5 expressional ratio as an adverse prognostic marker in colon cancer patients. Conclusions. The tumor progression-associated transcription factors STAT3, STAT4, and STAT5 are differently expressed in colon carcinoma tissue and colon mucosa. Moreover, the STAT3/STAT5 expression ratio is an independent prognostic marker in colon cancer patients. Colorectal cancer is one of the most common malignancies and the second leading cause of cancer-related mortality in the Western world.1,2 About 15–20 % of the patients reveal synchronous liver metastases at initial staging; additionally, 20–25 % develop metachronous liver metastases during the course of disease.3–5 The presence of liver metastases is associated with severely impaired 5-year survival, and liver resection still offers the only curative treatment option.6–8 Colon carcinogenesis entails the accumulation of multiple genetic aberrations.9,10 Another important step for initiating tumorigenesis, invasiveness, and metastatic spread, not only in colon cancer, is epithelial–mesenchymal transition.11–14 Moreover, the tumor microenvironment is known to dynamically interact with cancer cells and promotes cancer growth through releasing cytokines and growth factors.15 The STAT (signal transducer and activator of transcription) family of transcription factors consists of several proteins including STAT 1, 3, 4, and 5.16 The Janus kinase (JAK)/STAT signaling pathway is implicated in a variety of cancer entities, including ovarian carcinoma, rhabdomyosarcoma, osteosarcoma, and colon cancer.16–18

F. Klupp et al.

Ligand binding initiates an activation cascade, ultimately resulting in phosphorylation of STAT proteins. Phosphorylation enables STATs to translocate from the cytoplasm into the nucleus and to become transcriptorily active.19,20 Key activators of STAT proteins are cytokines like interferons and interleukins, as well as growth factors like transforming growth factor (TGF) b or oncogenes like SRC.21–23 Recent evidence suggests a role of STAT transcriptions factors in colorectal cancer progression. Indeed, STAT1, STAT3, and STAT5 are reportedly expressed in colorectal cancer and STAT4 in rectal cancer.17,20,24 Some studies describe correlations with patient survival.24,25 However, only few studies have evaluated the quantitative expression of various STAT proteins in a homogenous collective of colon cancer patients, with correlation to metastasis and overall survival. Here, we applied multiplex-based Luminex technology to analyze the specific expression of transcriptorily active STAT family members in colon cancer tissue and corresponding healthy colon mucosa from 104 patients and investigated putative correlations with patient prognosis.

mucinous tissue was separated from cancer tissue. Tissue lysis was performed with Procarta Transcription Factor Assay Whole Cell Lysis Kits (Affymetrix, Santa Clara, CA, USA) according to manufacturer’s protocol. Total protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) and Infinite 200 PRO Reader (Tecan Group, Ma¨nnedorf, Switzerland). Magellan Data Analysis Software (Tecan Group) was applied for data analysis. All samples were diluted to a concentration of 0.5 lg/ll of total protein. Luminex-Based Multiplex Assay Tissue lysates were processed using Procarta Transcription Factor Assay Kits (Panomics/Affymetrix) according to the manufacturer’s protocol. The Procarta Transcription Factor Assay kits were individualized for expressional analysis of the activated forms of the transcription factors STAT1, STAT3, STAT4, and STAT5. Semiquantitative mean fluorescence intensities of these factors were detected by Luminex 100 reader (Luminex, Austin, TX, USA).

MATERIAL AND METHODS Immunohistochemistry Patients Tissue samples of primary colon adenocarcinoma and of healthy colon mucosa from a series of 104 patients were included. All patients underwent surgery at the Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Germany, between September 2007 and March 2012. Written informed consent of all patients was obtained. The study was approved by the local ethics committee. Clinical characteristics like gender, date of birth, age at surgery, tumor location, histopathologic diagnosis including tumor, node, metastasis classification system, and International Union Against Cancer (UICC) stage, R classification, grading, adjuvant chemotherapy, and postoperative complications like anastomotic leakage and overall survival (time from operation up to death or last follow-up) were obtained from each patient. Tissue samples of adenocarcinoma of the colon were excluded if localized in the rectosigmoid closer than 16 cm to the anus. Tissue Material and Preparation Tissue was taken immediately after resection and stored at -80°C until further use. Healthy colon mucosa tissue was acquired at least 10 cm away from the tumor. Frozen sections (18 lm) were cut using a cryostat (CM 30509; Leica Microsystems, Wetzlar, Germany) and stained with cresyl violet. Optical light microscopy was applied to evaluate tissue quality, and remaining stromal or

Tissue samples were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the ethics committee of Heidelberg University. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissues samples for activated STAT3 (STAT3 antibody, phospho, ab76315; Abcam, Cambridge, UK) and activated STAT5 (STAT5 antibody, phospho, ab 32364; Abcam) was performed using the Dako autostainer with 1:100 dilution. Beforehand, slides were pretreated at pH 9.0. Evaluation was performed by a board-certified pathologist from the Institute of Pathology, University of Heidelberg. Western Blot Analysis Protein lysates were extracted from fresh frozen tissue samples using radioimmunoprecipitation assay buffer with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After measuring protein concentration with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), 30 lg protein were added to 6 9 Laemmli buffer (BioRad, Hercules, CA, USA), denaturated, and loaded to a 7.5 % gel. After electrophoresis, gels were blotted to polyvinyl difluoride transfer membrane (Millipore, Bedford, MA, USA), blocked, and incubated with primary (dilution 1:1000, and Tubulin antibody; Cell Signaling

STAT3/STAT5 Prognostic Marker in Colon Cancer

Technology, Danvers, MA, USA) and secondary antibodies (goat anti-rabbit HRP; Santa Cruz Biotechnology, Dallas, TX, USA; dilution 1:2000). Chemiluminescence and detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and a CCD camera. Data analysis was performed using ImageJ software 1.48 (National Institutes of Health, Bethesda, MD, USA) and standardized to Tubulin. Statistical Analysis Statistical analyses were conducted with Excel 2010 (Microsoft, Redmond, WA, USA) and SPSS version 21 (IBM, Armonk, NY, USA). The Wilcoxon signed rank test was used to determine expressional differences in different tissues. Expressional data are presented as mean ± SEM. Expressional changes in M1-stage tumors were assessed by the Mann–Whitney U test. The Kaplan-Meier method was used to estimate cancer-related survival. Differences between survival curves were evaluated by log rank test. The Cox proportional hazard model was applied to calculate survival related hazard ratios. Multivariate analysis was performed by Cox proportional hazard regression including the following covariates: age, UICC stage, R status, presence of liver metastases, chemotherapy, and expressional ratio of STAT3/STAT5 and STAT3/STAT4 in tumor tissue versus healthy colon mucosa. Results were considered significant at a p value of less than 0.05. RESULTS

TABLE 1 Correlation of clinical parameters of 104 patients with overall survival Characteristic

n

Mean overall survival (mo)

Gender Male

0.288 66

41.1

38

48.0

\median = 65 years

50

39.8

Cmedian = 65 years

54

43.3

Primary stoma

7

22.66

Secondary stoma

8

37.45

No stoma

89

44.24

Yes

8

49.2

No

96

42.8

Female Age at operation

0.775

Stoma formation

0.362

Anastomotic leakage

0.732

Operative revision

0.101

Performed

11

33.9

Not performed

93

44.43

T stage

0.291

T1 T2

2 11

NA 39.0

T3

69

44.4

T4

22

35.0

N0

44

49.3

N1

34

37.6

N2

26

36.6

M0

67

46.8

M1

37

34.5

Yes

37

34.7

No

67

50.7

R0

85

45.7

R1 R2

5 1

30.5 14.7

Rx

13

27.1

Yes

63

38.6

No

41

47.3

N stage

0.019

M stage

Patient Characteristics A total of 104 patients with adenocarcinoma of the colon were included in the study Table 1). Median age at the time of operation was 65 years. The median follow-up time was 729 ± 470 days (range 6–1980 days). Thirty-six patients died during follow-up. Mean overall survival of all patients was 42.6 months. Each patient underwent colon cancer surgery according to the localization of the tumor: 35 patients had right colectomy, 15 extended right colectomy, 3 extended left colectomy, 15 left colectomy, 21 sigmoidectomy, 4 proctosigmoidectomy, 6 high anterior resection, 1 low anterior resection, 1 transversectomy, and 3 subtotal colectomy. Detailed patient characteristics are outlined in Table 1. Patients with a positive lymph node status (N1, N2) or with distant metastases (M1) had a significantly reduced overall survival. Patients’ resection status correlated significantly with shortened overall survival. Moreover, adjuvant chemotherapy significantly correlated with shortened overall survival (Table 1).

p

0.002

Occurrence of liver metastases

0.002

Resection margin status

0.008

Adjuvant chemotherapy

0.036

NA not available

Expression of Individual STATs in Colon Cancer Tissue and Corresponding Healthy Mucosa The expression of the activated forms of the following STAT transcription factors was measured in colon cancer

F. Klupp et al.

STAT3

B

150

Fluorescence intensity

* 75

STAT5 **

60

30

0

uc os a

or Tu m

uc os a

M

Tu m

or

0

M

Fluorescence intensity

A

FIG. 1 Expression analysis of STAT proteins in colon cancer tissue and corresponding healthy colon mucosa. Expression of a STAT3 was reduced, whereas expression of b STAT5 was elevated in colon cancer tissue compared to corresponding healthy mucosa. Bars indicate mean fluorescence intensities ± SEM. *p \ 0.02; **p \ 0.001, n = 104 patients

STAT3/STAT5

**

Fluorescence intensity tumor/mucosa

100 80 60 40 20 0

M0

M1

FIG. 2 Expressional changes of STAT proteins in patients with distant metastases (M1) and no distant metastases (M0) at initial staging. Expressional ratio of STAT3/STAT5 was significantly elevated in M1 compared to M0 patients. Box plots represent median expression quotient, interquartile range, and total range. **p \ 0.001; *outliers, M0: n = 67 patients, M1: n = 37 patients

samples and corresponding healthy colon mucosa: STAT1, STAT3, STAT4, and STAT5. Contrary to the other STAT transcription factors, we could not observe a significant difference in the expression of STAT1 in colon cancer tissue and corresponding colon mucosa tissue (Supplementary Fig. 1a). STAT3, however, displayed a significantly lower expression in cancerous tissue (Fig. 1a). In contrast, STAT4 (Supplementary Fig. 1b) and STAT5 (Fig. 1b) both showed a significantly higher expression in colon cancer tissue compared to corresponding healthy colon mucosa. Collectively, these results suggest that colorectal tumors tend to up-regulate the expression of STAT4 and STAT5 but to down-regulate the expression of the STAT3 transcription factor.

Immunohistochemistry was applied to assess spatial expression patterns of STAT3 and STAT5 in primary colon tumors and corresponding colon mucosa. Both activated STAT3 and activated STAT5 were expressed in tumor cells (Supplementary Fig. 2a, b). Colon mucosal cells displayed weak expression of STAT3 (Supplementary Fig. 2c), whereas STAT5 was not detectable by immunohistochemistry in normal colon mucosa (Supplementary Fig. 2d). Thus, the quantitative expression values as delineated by Luminex technology seem to reflect the expression of STAT3 and STAT5 in tumor cells rather than in stromal cells or immune cell infiltrates (Supplementary Fig. 2). Correlation with M Stage In an initial attempt to investigate putative effects of these expressional changes on patient prognosis, we analyzed whether the expression of the individual STAT proteins was altered in patients who had distant metastases at the time of diagnosis (M1) compared to those in whom no distant metastases were found at initial staging (M0). As a measure of STAT protein expression, we calculated a quotient of the fluorescence intensities (indicating the activated forms of STAT proteins) in colon cancer tissue and corresponding healthy colon mucosa. We then analyzed differences between the M0 group and the M1 group of patients. The expression quotients of the transcription factors STAT1 and STAT3 were significantly higher in specimens of patients with disseminated disease (Supplementary Fig. 3a, b) than in the M0-staged patients. Surprisingly, the other two members of the STAT family showed reverse expression patterns. Indeed, the expression quotients for STAT4 and STAT5 proteins were significantly lower in M1-staged patients than in M0-staged patients (Supplementary Fig. 3c, d). These expressional differences became most striking when calculating a STAT3/STAT5 expressional ratio, which was severely increased in M1-staged patients compared to patients without distant metastases (Fig. 2). The expressional STAT3/STAT4 ratio was likewise significantly elevated in M1-staged patients (Supplementary Fig. 3e). Western blot analysis was performed as an alternative experimental readout to validate the observed expressional changes of STAT3 and STAT5 proteins in primary tumors from M0 versus M1 staged patients. Consistent with the outlined data obtained by Luminex technology, immunoblotting revealed a significantly lower STAT3/STAT5 expressional ratio in M0-staged patients than in M1-staged patients (Supplementary Fig. 4).

STAT3/STAT5 Prognostic Marker in Colon Cancer

Impact of STAT Expression on Survival We likewise associated the expressions of STAT1, STAT3, STAT4, and STAT5 with patient survival. For this purpose, the quotient of STAT expression in colon cancer tissue versus expression in corresponding healthy colon mucosa was calculated, and tumor/mucosa expression ratios of \2 and C2 were then correlated with overall survival.26–28 In case of STAT1 and STAT3, an expression at least twice as high in colon cancer tissue as in corresponding colon mucosa tissue correlated with significantly impaired overall survival (Supplementary Fig. 5a, b). Conversely, a STAT4 or STAT5 expression quotient tumor/mucosa of two or more was associated with significantly prolonged overall survival (Supplementary Fig. 5c, d). When calculating an expressional STAT3/STAT5 ratio, it became apparent that overall survival was strikingly impaired in

STAT3/STAT5

Overall survival [%]

100 80

tu/muc < med.

60 40 20

tu/muc ≥ med. P=0.002

0 0

1

2

3

4

5

Time after operation [years] FIG. 3 Correlation of STAT protein expression quotients (tumor/mucosa) and overall survival in univariate analysis. Expression ratio tumor/mucosa of [2 was defined as increased expression. An expressional ratio of STAT3/STAT5 (p = 0.002; hazard ratio 3.11; 95 % confidence interval 1.46; 6.65) higher than the median (med.) elicits significantly shortened overall survival

patients with a STAT3/STAT5 ratio higher than the median (Fig. 3). The same held true for an elevated expressional STAT3/STAT4 ratio, which was likewise associated with impaired survival (Supplementary Fig. 5e). However, multivariate analysis revealed that only an increased STAT3/STAT5 in tumor tissue versus healthy mucosa is an independent prognostic marker for impaired overall survival in colon cancer patients (Table 2). DISCUSSION STATs comprise a family of transcription factors, which are, along with their downstream targets, substantial regulators of carcinogenic processes like proliferation, differentiation, and apoptosis as well as metastasis formation.16,29,30 Indeed, numerous studies revealed aberrant expression of the STAT proteins in various cancers.18,30–32 In the present study, we evaluated the expression patterns and prognostic relevance of activated STAT1, STAT3, STAT4, and STAT5 in a homogenous collective of 104 patients with adenocarcinoma of the colon applying Luminex technology, which is characterized by high accuracy and reproducibility.33–36 Additionally immunohistochemistry was applied to investigate the spatial expression of STAT3 and STAT5 in colon carcinomas. It has been previously demonstrated that activation of STAT1 supports migration and chemoresistance of colon cancer cells, which is consistent with functional genetic studies revealing tumor-promoting functions of STAT1.37,38 Conversely, however, Simpson et al. found that high nuclear expression of STAT1 coincides with elevated numbers of intratumoral T cells and is associated with favorable prognosis in colorectal cancer.39 In the present study, activation of STAT1 in colorectal tumors was associated with metastatic spread and impaired overall survival. Similarly, protumorigenic effects have been linked to STAT3. Activation of STAT3 via oncogenic pathways involving TGF-b or the epidermal growth factor receptor mediates epithelial–

TABLE 2 Multivariate analysis (Cox proportional hazard regression model) of prognostic parameters for overall survival in colon cancera Characteristic

HR

95 % CI of relative risk

p

Median age

1.025

0.508–2.067

0.946

UICC stage

1.661

0.759–3.634

0.204

R stage

1.092

0.982–1.214

0.104

Adjuvant chemotherapy

0.536

0.151–1.905

0.335

Presence of liver metastases

1.519

0.475–4.863

0.481

STAT3/STAT4 expression ratio tumor tissue vs. healthy colon mucosa

0.311

0.061–1.583

0.159

STAT3/STAT5 expression ratio tumor tissue vs. healthy colon mucosa

4.468

1.051–18.994

0.043

HR hazard ratio, CI confidence interval, UICC International Union Against Cancer a

A high STAT3/STAT5 tumor/mucosa expressional ratio is an independent prognostic marker in colon cancer patients

F. Klupp et al.

mesenchymal transition, thus facilitating metastatic spread of colon cancer.16,22,40–42 In addition, STAT3 can support tumor spread via its immunomodulatory functions. For instance, interaction of NF-jB with STAT3 coregulates various inflammatory mediators with oncogenic functions.43–47 Further, STAT3-mediated expansion of regulatory T cells is required for the development of specific T helper cells which are able to promote tumor growth.47–50 Consistently, we found that increased activation of STAT3 is significantly associated with poor overall survival and metastatic spread. Intriguingly, controversial clinical evidence has been reported concerning the impact of STAT3 on survival. On the one hand, and in accordance with our results, Kusaba et al. associated elevated STAT3 expression in colorectal cancer samples with enhanced tumor infiltration, lymph node metastases, and poor prognosis.25,51 Congruently, a large study based on tissue microarrays revealed that patient survival was specifically impaired upon tumoral overexpression of activated STAT3.38 Park et al. found that STAT3 expression in colorectal carcinoma tissue samples correlated with the occurrence of distant metastases, and various other studies similarly evince a tumor-promoting role of STAT3 in colorectal cancer.17,32,52–54 On the other hand, applying immunohistochemical analysis of tissue microarrays, Gordziel et al. identified STAT3 as a favorable marker in colorectal carcinoma.24 This heterogeneity of data concerning the prognostic impact of STAT3 expression in colorectal cancer might be partly attributable to diverse technical methodologies applied in different studies or to small case numbers in some of these individual reports.25,32,51 Taken together, our data revealed that activated STAT1 and STAT3 exert tumorpromoting functions in colorectal cancer. Although conflicting evidence likewise exists, these findings are consistent with a majority of available reports reflecting the biologic and prognostic significance of STAT1 and STAT3 in human colorectal cancer.17,37,55 In striking contrast to these assumed tumor-promoting functions of STAT1 and STAT3, our study indicated that activation of STAT4 and STAT5 exerts antitumorigenic effects, resulting in reduced metastatic spread and favorable patient prognosis. Although less is known concerning specific functions of STAT4 in colorectal cancer, tumorsuppressive effects of unphosphorylated STAT5 have been demonstrated in colon cancer cells in vitro and in mouse xenograft models in vivo.56 However, isoform-specific protumorigenic effects of STAT5 have likewise been documented, and in colorectal cancer, the expression of activated STAT5 in colorectal cancer specimens has been linked to impaired survival.47,57–60 It is thus conceivable that STAT5 (and other STAT proteins) can exert tumor-promoting as well as tumor-suppressive functions, depending on the cellular tumor microenvironment, the availability of molecular downstream targets, and the level of STAT

activation.17,30,47,56,61 It is in this context remarkable that loss of STAT5 may enhance cancer development through activation of TGF-b and STAT3, indicating an interplay between different STAT proteins in tumor spread.62 Given their diverse and nonredundant functions in colorectal cancer progression, it seems to be worthwhile to simultaneously assess the expression of various STAT proteins when attempting to apply them as a prognostic factor in individual colorectal cancer patients. Indeed, we were able to demonstrate that only a STAT3/STAT5 expressional ratio could serve as an independent predictor for survival in multivariate analyses, whereas the expression of either STAT alone could not. Therefore, the balance of the various STAT proteins seems to be a relevant factor in determining the prognostic effect for the patient. Immunohistochemical staining revealed that activated STAT3 and STAT5 protein localized predominantly to tumor cells. Nonetheless, we cannot exclude the notion that stromal cells likewise express STAT proteins, enabling cross-talk between STAT-expressing tumor cells and tumoral stroma cells, which might in turn mediate the tumor-promoting effects of the tumor microenvironment, as previously described.15,38,63 In summary, our data encourage a significant role of STAT1, STAT3, STAT4, and STAT5 in progression and metastasis of colon carcinoma, with important implications for overall survival. Among the major findings, our results indicate that the predictive value of STAT expression can be fortified when comparing individual expressions of various STAT proteins. Indeed, we demonstrate for the first time that an expressional STAT3/STAT5 ratio qualifies as an independent marker for impaired prognosis. On the basis of our present results, we assume that it is necessary to determine the expressional pattern of various STAT proteins—especially STAT3 and STAT5—and their activation level in order to enable a predication on prognosis. The present study thus specifies the predictive value of STAT proteins as prognostic factors in human colon carcinoma and encourages the concept that these molecules represent important prognostic factors and putative molecular targets for tailored anticancer therapy. ACKNOWLEDGMENT We thank Tina Lerchl, Gloria Laukemper, and Sascha Hinterkopf for their technical support. Supported in part by Clinical Research Unit KFO 227, ‘‘From Primary Tumor Progression toward Metastases,’’ funded by the German Research Foundation (DFG). DISCLOSURE

The authors declare no conflict of interest.

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STAT5 Ratio is an Independent Prognostic Marker in Colon Carcinoma.

Signal transducer and activator of transcription proteins (STATs) are crucial regulators of cell growth and differentiation; however, their specific p...
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