Carboxypeptidase E mRNA: Overexpression predicts recurrence and death in lung adenocarcinoma cancer patients

1. Introduction

Lung cancer is the leading cause of cancer death in both men and women, worldwide. In the United States, lung cancer currently accounts for 24% of cancer deaths and is on the rise [1]. According to the American Cancer Society’s estimates in USA, there will be about 235,760 (119,100 in men and 116,660 in women) new cases of lung cancer diagnosed in 2021 and 131,880 deaths, with the risk being much higher for smokers than non-smokers [2]. Lung cancer has been characterized into two main groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), each accounting for 13% and 84% respectively. NSCLC consists of three main subtypes based on histology: adenocarcinoma, squamous cell carcinoma and large cell carcinoma [3]. Depending on the stage of the disease, various types of treatments have been used. For lung adenocarcinoma (LADC), the most common is surgery followed by tyrosine kinase inhibitors (TKIs) target therapy and radiation therapy. Despite successful treatments, especially with improved drugs for TKIs therapy, some LADCs will recur, either locally, or as distant metastasis, which is the major cause of mortality [4]. Thus, identification of additional biological markers than epidermal growth factor receptor (EGFR) mutations that could predict future recurrence of the tumor is of extreme importance, as that will prompt more vigilant follow-up of high risk patients for early detection of recurrence which will improve outcome and survival.

Carboxypeptidase E (WT-CPE) and a 40 kD N-terminal truncated splice variant of CPE, (CPE- Δ N, Accession # MT035840) are expressed in human cancer cells and have been reported to be a good prognostic biomarker for hepatocellular carcinoma (HCC) [5, 6], colorectal cancer [7], glioblastoma [8], and cervical cancer [9]. A study of 95 LADC patients showed that expression of CPE- Δ N protein, a N-terminal truncated form of CPE, was specifically in the tumor and not in the pericarcinoma, and was associated with poor prognosis [10].

CPE was originally discovered as a prohormone processing enzyme and is expressed in endocrine organs and brain [11, 12], and thus its expression in endocrine and neuroendocrine tumors is not surprising. In cancer cell lines, overexpression of wild-type (WT) CPE has been demonstrated to enhance proliferation in a colorectal cancer cell line [13], and glioma cell lines [8]. Additionally, experiments where recombinant WT-CPE has been added to the culture media of HCC cells under hypoxic conditions have demonstrated that it functions as a survival factor through activating the ERK signaling pathway and enhancement of expression of the mitochondria pro-survival protein, BCL2 [14]. Secreted WT-CPE in media of glioblastoma cell lines [8] or addition of recombinant WT-CPE exogenously to, fibrosarcoma (HT1080) cells showed no effect on promoting cell migration or invasion [14]. However, N-terminal truncated forms of CPE, (CPE- Δ N), when transfected into a HCC cell line [5], Panc-1 cells [15] and osteosarcoma Saos-2 cells [16], promoted migration and invasion, as well as proliferation. Treatment of various cancer cell lines, including those derived from pancreas [17], and osteosarcoma [18] with CPE siRNA that suppresses expression of both CPE- Δ N and WT-CPE mRNA indiscriminately, inhibited proliferation and invasion. Thus loss-of-function and gain-of-function studies indicate that CPE promotes cell proliferation, migration and invasion to drive tumor growth and metastasis in different cancer types.

Quantitative RT-PCR, mRNA expression profiling and immunostaining studies have detected CPE expression in SCLC [19, 20] and NSCLC [21], including a subclass of LADC [22], although the exact form(s) of CPE mRNA and protein present in those tumors were not characterized. Here, we have evaluated whether CPE/CPE- Δ N mRNA could serve as a biomarker for predicting recurrence and survival in LADC patients. We have analyzed a cohort of 86 LADC patients and determined the levels of CPE/CPE- Δ N mRNA in the resected tumors. Our results indicate that high levels of CPE/CPE- Δ N mRNA in LADC are significantly associated with recurrence and poor survival of patients at all stages of the disease.

2. Materials and methods

Our previous study quantifying CPE/CPE- Δ N mRNA levels by qRT-PCR in primary HCC tumors (T), versus surrounding tissue (N) revealed that a ratio of T/N $>$ 2 predicted future development of recurrence [6]. In another large study of 219 colorectal cancer patients, high levels of CPE- Δ N mRNA expression were closely correlated with tumor differentiation, recurrence and lymph node metastasis [7]. More recently, it has been reported that overexpression of CPE/CPE- Δ N protein detected both in the nuclei and cytoplasm is correlated with pelvic lymph node metastasis and poor prognosis in patients with early-stage cervical cancer [9]. These clinical studies indicated that CPE/CPE- Δ N is a potentially powerful prognostic biomarker for predicting recurrence and metastasis in many cancer types. Here, we have evaluated whether CPE/CPE- Δ N mRNA, could be a useful prognostic biomarker for predicting recurrence and death in lung adenocarcinoma patients. In this study, we showed that in NSCLC of the adenocarcinoma subtype, high levels of CPE/CPE- Δ N mRNA expression predicted recurrence and death in these patients. This is consistent with a mRNA expression profiling study which revealed that high CPE mRNA expression in a subclass of lung adenocarcinoma indicated poor prognosis [22]. Our study in lung adenocarcinoma patients revealed a sensitivity of 84.8% for CPE/CPE- Δ N mRNA as a biomarker for predicting recurrence, and 90.3% for predicting death. The specificity for this biomarker for predicting recurrence and death was 35.0% and 32.7% respectively. Thus our data indicate that CPE/CPE- Δ N mRNA is a potentially useful prognostic biomarker with high sensitivity for lung adenocarcinoma patients. Our study with up to 7-year follow-up of patients post-operatively corroborates and extends a previous small study done by another group showing that patients with adenocarcinoma expressing CPE- Δ N proteins which included several immunostained bands smaller in size than WT-CPE, versus those without CPE- Δ N expression analyzed by Western blot, had a significantly higher recurrence and metastasis rate over a 3 year follow-up period post-operatively [10]. Subsequently, the same group demonstrated that immunostaining intensity of nuclear CPE in 195 lung adenocarcinoma clinical samples increased with increasing TNM stage I, II, III, although no other analysis was done [25]. There are no specific antibodies that would cross react with CPE- Δ N specifically and not WT-CPE. Hence the nuclear CPE immunostaining in the those LADC samples likely represents CPE- Δ N since subcellular fractionation followed by Western blot, and transfection studies in the literature on different cancer cell lines have specifically shown localization of CPE- Δ N in both cytoplasm and nucleus, while WT-CPE is only in the cytoplasm [5, 15]. Thus scoring nuclear CPE immunostaining in tissue microarray and paraffin sections for quantifying CPE- Δ N expression, can apparently provide an indication of poor prognosis, if immunostaining of nuclear CPE in the tumor is intense. However, for the purpose of biomarker development, our study using a CPE/CPE- Δ N mRNA PCR assay provides a more quantitative high throughput measurement than immunohistochemistry. The combination of both the CPE/CPE- Δ N mRNA PCR assay and scoring of nuclear CPE immunostaining intensity could render CPE a very useful biomarker for predicting recurrence, metastasis and death in LADC.

In our analysis of the time to recurrence, one of the most important findings of this study is that high CPE/CPE- Δ N mRNA copy numbers ( $>$ 10000) predicted recurrence in the cohort of patients with stage I and II adenocarcinoma, with most recurrence within 20 months after resection (Fig. 1a, 2). Currently, there are no good prognostic biomarker that could identify high risk stage I and II lung adenocarcinoma patients. Thus, CPE/CPE- Δ N mRNA is potentially a useful molecular biomarker with high sensitivity for early diagnosis of high risk patients who would rapidly develop recurrence, even though the histopathology of their tumor based on staging (I and II) would have indicated good prognosis. Such knowledge would help prompt more frequent follow up for these high risk patients and determine the strategies for treatment post-surgery that would improve outcome. There are a number of molecular biomarkers for NSCLC using microarray and proteomics technology to identify genetic mutations (for review see [26]. These include K-ras, p53, EGFR [27, 28, 29]; hypermethylation of genes (e.g. aberrant p16 methylation) [30]; or increased or decreased expression of proteins (e.g. hsp70 and Rab37, CYP3A4 and CYP3A5) [31, 32] that characterizes aggressive tumors and poor prognosis, but they do not predict recurrence of Stage I or Stage II patients. Additionally, serum/plasma biomarkers for NSCLC have also been reported such as GM2-activator protein [33], plasma D-dimer [34] and mRNA-21 [35]. Bioinformatic analyses of databases have identified IncRNAs as potential predictive biomarkers of lung adenocarcinoma [36]. As well, circulating microRNAs and circRNA, free or in exososomes, ctDNA and circulating tumor cells are emerging as possible biomarkers in diagnosis and quantification of lung cancer burden [37, 38]. However, none of these potential biomarkers have been translated to clinical use yet. As commonly seen in biomarker research, one biomarker alone may not produce high enough prediction accuracy; therefore, larger-scale future research is warranted in combining CPE/CPE- Δ N mRNA and protein with other existing and novel biomarkers, as well as other risk factors, to improve the risk prediction model of adenocarcinoma recurrence and death.

Subgroup analysis showed another interesting finding in that females with high copy numbers of CPE/CPE- Δ N mRNA were associated with higher recurrence and death. Furthermore, non-smokers that have copy numbers of CPE/CPE- Δ N above the cutoff of 10,000 were also highly associated with recurrence and death. The correlation may be due in part to the fact that the female lung cancer patients were mostly non-smoking patients in our recruited Asian patient cohort. We should emphasize that the subgroup analyses were based on relatively small sample size, and mostly exploratory and hypotheses-generating. Larger studies are needed to replicate and to confirm these findings, so that they could be translated into the population level. Nevertheless, these findings motivate further investigations into the relationship between CPE/CPE- Δ N mRNA and cancer progression among women, non-smokers and early and late stage patients with lung adenocarcinoma.

In conclusion, we have provided evidence that CPE/CPE- Δ N mRNA is a potential candidate biomar-ker for predicting recurrence and death in lung adenocarcinoma patients, and is especially clinically useful for identifying high risk patients very early when the disease is at stage I and II. Moreover, non-smokers are also at high risk of recurrence and death when the CPE/CPE- Δ N mRNA levels are high. Of particular significance is that this study has set the stage for future large scale exploration to confirm the potential utility of CPE/CPE- Δ N mRNA as a biomarker to select early stage lung adenocarcinoma patients for adjuvant chemotherapy or extended adjuvant therapy post surgery.

Author contributions

Conception: YPL, Y-CW.

Interpretation or analysis of data: I-YK, DL, W-WL, YPL, Y-CW.

Preparation of the mauscript: I-YK, DL, YPL, Y-CW.

Revision for important intellectual content: I-YK, DL, YPL, Y-CW.

Supervision: YPL, Y-CW.

Supplementary data

The supplementary files are available to download from https://dx-doi-org.web.bisu.edu.cn/10.3233/CBM-210206.