Introduction
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide, with an estimated 1.4 million cases and 693,900 deaths occurring in 2012 [
1]. Although the incidence of CRC in high-risk/high-income countries has varied over the past 20 years, it is increasing in certain historically low-risk countries, such as Spain and several countries in Eastern Europe and East Asia, which have been ascribed to changes in dietary patterns and risk factors toward a so-called Western lifestyle including unhealthy diet, obesity, and smoking [
2‐
5]. In addition, increases in CRC mortality rates have been observed in countries that have more limited resources and increasing incidence [
6‐
8]. At present, classification according to tumor–node–metastasis (TNM) stage provides valuable prognostic information and guides therapy decisions for CRC patients; nevertheless, clinical outcome differs greatly even among patients of the same TNM category [
2]. Thus, there is an urgent need to search for valuable biomarkers to improve prognosis prediction and clinical outcome of patients with CRC.
Dehydrogenase/reductase (SDR family) member 9 (DHRS9), also known as retinol dehydrogenase L (RDHL), has been identified as a member of the short-chain dehydrogenases/reductase (SDR) family that converts retinol to retinal. Previous studies demonstrated that DHRS9 is involved in the biosynthesis of all-trans-retinoic acid (atRA), which displays important anti-tumor activity through inhibition of cell proliferation, induction of cell differentiation, and apoptosis and has been used in several cancer therapies including acute promyelocytic leukemia, squamous cell carcinoma, neuroblastoma, and hepatocellular carcinoma [
9‐
13]. The enzymatic activity of DHRS9 was firstly characterized by Soref et al. in airway epitherial cells (referred to as hRDH-TBE in their publication) [
14]. Later, Jette et al. reported that DHRS9 messenger RNA (mRNA) was primarily expressed in the colon, and its expression was low but detectable in the heart, spleen, placenta, and lung [
15]. Using microarray and reverse transcriptase-PCR analysis, they demonstrated that DHRS9 was frequently and significantly downregulated in colon adenomas and carcinomas as compared with normal colon tissues. Consistent with the data from the adenoma and carcinoma samples, they found that colon-cancer-derived cell lines expressed low or undetectable levels of DHRS9 and displayed poor conversion of retinol to retinoic acid when compared to normal epithelial cells. Recently, a significant decreased expression of DHRS9 mRNA in CRC has also been confirmed by Kropotova et al. [
10]. Mechanistic investigations revealed that reintroduction of the tumor suppressor adenomatous polyposis coli (APC) into the APC-deficient colon carcinoma cell line HT29 resulted in increased mRNA expression of DHRS9 via the transcription factor CDX2 [
15]. In addition, DHRS9 could be induced by the lytic Epstein–Barr virus (EBV) immediate–early protein, BZLF1, in AGS gastric carcinoma cells containing the lytic form of EBV infection and EBV-positive Burkitt lymphoma cells [
9]. Since lack of retinoic acid biosynthesis has been proposed as a mechanism contributing to the development of colon adenomas and carcinomas, we hypothesized that dysregulation of DHRS9 expression may be associated with aggressive clinical behavior of CRC. However, the clinical relevance of DHRS9 expression in CRC has not been investigated.
In the present study, we detected both the mRNA and the protein expression levels of DHRS9 in CRC clinical samples and further analyzed the correlation of DHRS9 expression with clinical features and with patient survival. Our results demonstrated that decreased expression of DHRS9 correlates with tumor progression and might serve as an independent unfavorable prognostic indicator for patients with CRC.
Discussion
In the present study, we reported that DHRS9 expression was both transcriptionally and translationally downregulated in primary CRC clinical tissues compared with corresponding adjacent normal mucosa tissues. In addition, reduced expression of DHRS9 was significantly correlated with a variety of important clinicopathologic parameters including lymph node metastasis, TNM stage, disease recurrence, and vital status. Finally, survival analysis revealed that lower DHRS9 expression in tumors significantly predicted poorer DFS and DSS and was an independent unfavorable prognostic indicator for CRC. Moreover, combination of DHRS9 with TNM stage had a better survival predictive power than either of the two parameters alone.
Decreased expression of DHRS9 has been documented previously by Jette et al. and Kropotova et al. at the mRNA level [
10,
15]. Our real-time qPCR data were also consistent with their findings. Of note, we observed a significant decrease in DHRS9 mRNA level (over twofold) in 65.5 % (38/58) of the cancerous specimens tested, while Jette et al
. reported that expression of DHRS9 was decreased at least twofold in 90 % (9/10) of carcinoma samples. This discrepancy may be attributed to the different ethnic groups studied, sample size, or endogenous control used for normalization. In addition, our subsequent Western blot assay on the same corresponding samples confirmed that DHRS9 protein was significantly downregulated in 74.1 % (43/58) of the cancerous tissues tested. Furthermore, immunohistochemical analyses of 163 paired CRC samples showed that the immunoreactivity of DHRS9 protein was observed in the cytoplasm, and 91.4 % (149/163) of the normal colorectal mucosa tissues tested were classified as DHRS9-positive, whereas only 57.7 % (94/163) of the cancerous samples were classified as DHRS9-positive. Among them, 46 % (75/163) of the normal mucosa tissues examined were moderate–strong staining of DHRS9 protein, while only 19 % (31/163) of the cancerous tissues showed virtually the same immunoreactivity. Thus, our results definitely confirmed the significant downregulation of DHRS9 expression in CRC at both the mRNA and the protein level. The reported induction of DHRS9 by the tumor suppressor gene APC [
15] may, at least partly, explain the reason why DHRS9 expression is reduced in CRC. Nevertheless, the detail mechanism for DHRS9 downregulation in CRC remains to be clarified.
Interestingly, according to our results, decreased expression of DHRS9 protein in CRC was significantly correlated with increased lymph node metastasis, advanced TNM stage, increased disease recurrence, and patient death, indicating that DHRS9 might be negatively involved in the progression of CRC. It is well established that atRA exerts multiple anti-tumor effects by inhibiting proliferation, reducing colony formation, blocking anchorage-independent growth, promoting differentiation, inducing apoptosis, and suppressing invasiveness of cancer cell [
12,
22‐
27], and significantly impaired atRA biosynthesis has been noted in a variety of human malignancies, such as prostate cancer, breast cancer, ovarian cancer, gastric cancer, and CRC [
15,
28‐
31]. Meanwhile, DHRS9 belongs to the atRA-generating enzymes [
13,
15,
32], and its reduced mRNA expression has also been observed in CRC tissues and CRC-derived cell lines [
10,
15]. Moreover, Jette et al. found that diminished atRA biosynthesis activity was accompanied with decreased DHRS9 expression in a number of CRC-derived cell lines and that reintroduction of the tumor suppressor APC into APC-deficient colon cancer cells not only induced DHRS9 expression but also increased the conversion of retinol to retinoic acid [
15]. Therefore, we hypothesized that DHRS9 might, at least partly, influence the progression of CRC through atRA-related pathway. To test this hypothesis, we detected the mRNA expression of DHRS9 and some downstream targets of atRA or CRC cell progression related genes. Wang et al. previously showed that atRA induces the tumor suppressor gene XAF1 expression through an interferon regulatory factor-1 element in colon cancer [
33], and Woo et al. recently reported that atRA induces expression of the tumor suppressor gene E-cadherin via inhibition of DNA methylation in HCT116 cells [
34]. Interestingly, we found that DHRS9 mRNA expression was positively correlated with XAF1 and E-cadherin mRNA expression in the 58 CRC samples tested (data not shown). These preliminary findings also support a tumor inhibitory role of DHRS9 in human CRC. Nevertheless, it should be noted that whether reduction of DHRS9 indeed contributes to the development and progression of CRC remains to be carefully determined, for the decrease in DHRS9 expression may also be the result of other factors that lead to cancer progression, rather than downregulation of DHRS9 leading to cancer progression. Further in vitro and in vivo studies using gain-of-function as well as loss-of-function strategies are warranted to address this issue.
Another interesting finding of the present study was the prognostic significance of DHRS9 in CRC. Decreased DHRS9 protein expression was significantly associated with shortened DFS and DSS of CRC patients. In univariate analysis, DHRS9 protein emerged as a significant prognostic factor of clinical outcome. Moreover, in multivariate analysis, DHRS9 still emerged as a significant independent predictor of survival in addition to tumor stage. Thus, to our knowledge, the current study is the first to report the prognostic value of DHRS9 in CRC. Notably, our results revealed that TNM stage also is a significant prognostic factor for CRC patients, which is consistent with the well-recognized adverse prognostic effect of tumor stage [
35] and confirms that our cohort was representative and that the survival analyses were valid (Table
3 and Fig.
3). More importantly, we demonstrated that combination of DHRS9 protein expression with TNM stage has a more powerful efficiency in prognosis prediction when compared to each of the two parameters alone. Currently, the underlying mechanism for the prognostic importance of DHRS9 expression in CRC is not known and requires further investigation; nevertheless, our data suggest that combination of DHRS9 with TNM stage may have a better prognostic value and could serve as a promising biomarker for classification of CRC patients into distinct risk subgroups and guide individualized therapy choices.
There were several limitations in the present study. Although our results revealed the clinicopathologic correlation and the prognostic value for DHRS9 expression in a cohort of CRC patients, they did not elucidate the role of DHRS9 expression in the development of CRC. Besides, due to the limitation of follow-up period, the median survival time of patients in the high DHRS9 expression group could not be obtained; thus, our current results could not accurately reflect the survival of patients in these subgroups. Additionally, the number of samples in the present study is still relatively small. Therefore, future multi-center, prospective studies using larger cohorts are necessary to verify the robustness of our findings before clinical translation.
In conclusion, we here provided the first evidence that DHRS9 protein expression was frequently downregulated in CRC tissues and that decreased expression of DHRS9 was significantly associated with disease progression and poor outcome of CRC patients. Our data suggest that DHRS9 could be used as a potential prognostic biomarker for CRC and that combination of DHRS9 with TNM stage or other parameters may enhance its performance in prognostic prediction. Apart from its prognostic value, results from the present study encourage further investigation of its potential role in CRC pathobiology.