AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report

Abstract

Purpose

Salivary gland cancer (SGC) is a rare and heterogeneous disease with significant differences in recurrence and metastasis characteristics. As yet, little is known about the mechanisms underlying the initiation and/or progression of these diverse tumors. In recent years, the AAA+ ATPase family members Pontin (RuvBL1, Tip49a) and Reptin (RuvBL2, Tip49b) have been implicated in various processes, including transcription regulation, chromatin remodeling and DNA damage repair, that are frequently deregulated in cancer. The aim of this study was to assess the clinical and functional significance of Reptin and Pontin expression in SGC.

Methods

Immunohistochemical staining of Pontin, Reptin, β-catenin, Cyclin D1, TP53 and MIB-1 was performed on a collection of 94 SGC tumor samples comprising 13 different histological subtypes using tissue microarrays.

Results

We found that Reptin and Pontin were expressed in the majority of SGC samples across all histological subtypes. Patients with a high Reptin expression showed a significantly inferior 5-year overall survival rate compared to patients with a low Reptin expression (47.7% versus 78.3%; p = 0.033), whereas no such difference was observed for Pontin. A high Reptin expression strongly correlated with a high expression of the proliferation marker MIB-1 (p = 0.003), the cell cycle regulator Cyclin D1 (p = 0.006), accumulation of TP53 as a surrogate p53 mutation marker (p = 0.042) and cytoplasmic β-catenin expression (p = 0.002). Increased Pontin expression was found to significantly correlate with both cytoplasmic and nuclear β-catenin expression (p = 0.037 and p = 0.018, respectively), which is indicative for its oncogenic function.

Conclusions

Our results suggest a role of Reptin and Pontin in SGC tumor progression and/or patient survival. Therefore, SGC patients exhibiting a high Reptin expression may benefit from more aggressive therapeutic regimens. Future studies should clarify whether such patients may be considered for more radical surgery, extended adjuvant therapy and/or targeted therapy.

Introduction

Salivary gland cancer (SGC) accounts for 3–6% of all head and neck cancers [1] and constitutes a heterogeneous group of malignant tumors originating from diverse anatomical sites, with various histological appearances and significant differences in their propensities to develop recurrences and metastases [2, 3]. The WHO classification system distinguishes 24 histological entities of malignant SGC tumors. Although studies dealing with the pathogenesis of some of the SGC subtypes have been reported [4–7], currently little is known about the mechanisms driving the initiation and/or progression of these diverse tumors. Primary surgery is the standard therapy for SGC whenever feasible, and adjuvant radiotherapy is particularly employed in patients with locally advanced tumors [8]. However, therapeutic options for patients with advanced disease and especially for those with metastatic disease are limited, and both chemotherapy and molecular targeted therapy regimens have been largely disappointing in these cases [9, 10].

Due to the extensive heterogeneity of these tumors, the dissection of histologic tumor appearances and molecular mechanisms driving SGC disease progression is of special interest. Until today, treatment strategies for SGC largely follow uniform procedures for all entities, whereas different subtypes may require individualized therapeutic regimens. Therefore, there is an urgent need for the identification of biomarkers related to clinical outcomes. A further understanding of the biology of these tumors may additionally lead to the identification of targets suitable for novel individualized therapies.

In recent years, the AAA+ ATPase (ATPases Associated with various cellular Activities) family members Pontin (RuvBL1, Tip49a) and Reptin (RuvBL2, Tip49b) have been implicated in various cellular processes, including transcription regulation, chromatin remodeling and DNA damage repair [11–14], which are frequently deregulated in cancer. Pontin and/or Reptin have, for example, been found to be highly expressed in colorectal and breast cancer cells as well as in renal cell carcinoma cells [15–19]. Moreover, in hepatocellular carcinoma high Reptin levels have been found to correlate with a poor clinical outcome [20]. Recently, it has been shown that targeting Pontin and/or Reptin may serve as an attractive novel strategy for cancer therapy, and the first inhibitors for Reptin and/or Pontin are already under way [21, 22].

Here, we describe differential Pontin and Reptin expression patterns in defined SGC subgroups and provide data on the clinico-pathological relevance of these two AAA+ ATPases. Furthermore, we assess putative associations of Reptin and Pontin expression with several proliferation and cell cycle control markers such as MIB-1 (Ki-67), Cyclin D1 and TP53. Based on previously reported associations between Pontin, Reptin and β-catenin expression [23, 24], as well as the fact that Cyclin D1 is a well-known transcriptional target of β-catenin [25], we also investigated β-catenin expression and its subcellular localization in relation to Pontin and Reptin expression.

Materials and methods

Patient samples and clinical data

The study was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. Written informed consent was obtained from each patient and the scientific protocol was approved by the local ethics committee (No. 2012–370-f-S). Samples were processed under pseudonyms. All experiments have been performed at the University Hospital of Münster. In a retrospective design, we analyzed SGC samples obtained at the time of surgical resection from 94 consecutive patients newly diagnosed with a primary parotid gland cancer. All patients were treated at the Department of Otorhinolaryngology, Head and Neck Surgery at the University of Münster between July 1988 and March 2014. The treatment modality employed was definitive surgery (histological subtypes and patient characteristics are shown in Tables 1 and 2).

Table 1.

Histopathological subtypes

Histology	n	%
Acinic cell carcinoma	13	13.8
Mucoepidermoid carcinoma	7	7.4
Adenoid cystic carcinoma	10	10.6
Cystadenocarcinoma	1	1.1
Low-grade cribriform cystadenocarcinoma	1	1.1
Salivary duct carcinoma	22	23.4
Adenocarcinoma NOS	6	6.4
Squamous cell carcinoma	10	10.6
Large-cell undifferentiated carcinoma	3	3.2
Carcinoma ex pleomorphic adenoma	15	16.0
Myoepithelial carcinoma	3	3.2
Basal cell adenocarcinoma	2	2.1
Sebaceous carcinoma	1	1.1
Total	94	100.0

Table 2.

Patients’ characteristics

Patients’ characteristics	N (%)
Patients	94
Male	49 (52.1%)
Female	45 (47.9%)
Age (years)
Mean ± SD	60.8 ± 2.0
Minimum/Maximum	11/90
Type of parotidectomy
Unknown	1 (1.1%)
Lateral	11 (11.7%)
Total	54 (57.4%)
Radical	27 (28.7%)
Subtotal	1 (1.1%)
Type of neck dissection
Unknown	15 (16.0)
None	9 (9.6%)
Selective	51 (54.3%)
Radical	19 (20.2%)
Radiotherapy postoperatively
Unknown	4 (4.3%)
Yes	57 (60.6)
No	33 (35.1)
Resection margins
R0	58 (61.7%)
R1	31 (33.0%)
Rx	5 (5.3%)
pT-stage
pTx	3 (3.2%)
pT1	27 (28.7%)
pT2	25 (26.6%)
pT3	25 (26.6%)
pT4a	11 (11.7%)
pT4b	3 (3.2%)
pN-stage
pNx	8 (8.5%)
pN0	48 (51.1%)
pN1	6 (6.4%)
pN2b	30 (31.9%)
pN2c	2 (2.1%)
cM-stage
cMx	34 (36.2%)
cM0	56 (59.6%)
cM1	4 (4.3%)
Lymphangiosis
Unknown	45 (47.9%)
Yes	27 (28.7%)
No	22 (23.4%)
Hemangiosis
Unknown	53 (56.4%)
Yes	16 (17.0%)
No	25 (26.6%)
Perineural invasion
Unknown	47 (50.0%)
Yes	27 (28.7%)
No	20 (21.3%)

The patient ages at diagnosis ranged from 11 to 90 years (mean age 60.8 ± 2.0 years); 49 (52.1%) of the patients were male and 45 (47.9%) were female. With the exception of four patients, all were free of distant metastasis (cM0) at the time of diagnosis as evaluated via computed tomography scans of the thorax and via abdominal ultrasonography. Follow-up data were collected at periodic visits with intervals of 3–6 months at the outpatient clinic of the Department of Otorhinolaryngology. The mean follow-up time was 58 months (range 0–191 months).

Pathological evaluations

Pathological evaluations were performed as described previously [4]. In brief, formalin-fixed and paraffin-embedded (FFPE) tissue samples of all cases were selected on the basis of routinely generated pathological reports at the time of first diagnosis. All cases were re-evaluated and the diagnoses were updated according to the WHO 2005 classification of tumours of salivary glands. Additionally, FISH analyses were performed in all cases for MAML, MYB and ETV-6 to uncover translocations, and the results were included in the differential diagnoses when appropriate. The ETV-6 positive cases, i.e., cases of the newly described entity of mammary-analogue secretory carcinoma [26], were included in the WHO category adenocarcinoma NOS. In the case of squamous cell carcinoma (SCC) a thorough clinical examination of the head and neck region and the skin was performed to rule out a primary site outside the salivary glands. Only SCC cases with tumor growth within the salivary gland parenchyma were included.

Tissue microarray construction, FISH and immunohistochemistry

From FFPE material of all included cases two core biopsies of 1 mm diameter out of the tumor area were taken to generate tissue microarrays (TMA). The exact site of biopsy was determined through microscopic evaluation of the carcinomatous area on corresponding H&E-stained slides. FISH analyses and immunohistochemical (IHC) staining were performed on TMA slides. FISH analyses for MYB, MAML and ETV-6 translocations were performed as described previously using locus-specific break-apart probes (ZytoVision GmbH, Bremerhaven, Germany) [27, 28]. Tumors with break-apart signals in at least 20% of the cells were considered translocation positive. IHC staining was carried out using a BenchMark ULTRA apparatus (Ventana Medical Systems, Inc., Tucson, USA) for Reptin and Pontin and a Dako autostainer (Dako Deutschland GmbH, Hamburg, Germany) for MIB-1, β-catenin, Cyclin D1 and TP53, according to the manufacturer’s instructions. TMA antigen retrieval was induced by heating in low or high pH buffer. For visualization, the Opti View DAB Kit (Ventana Medical Systems, Inc., Tucson, USA) was used for Reptin and Pontin, and the LSAB method with AP/RED (Dako Deutschland GmbH, Hamburg, Germany) for MIB-1, Cyclin D1 and TP53 and with HRP/DAB (Dako) for β-catenin. The following antibodies and concentrations were used: anti-Pontin (clone 5G3–11, 1:50), generated by the Huber laboratory, University of Jena [29], anti-Reptin (Tip49b, BD Transduction Laboratories, Heidelberg, Germany, 1:500), anti-MIB-1 (M7240, Ki-67, Dako, 1:100), anti-β-Catenin (RB-9035-P, Thermo Fisher Scientific Inc., Waltham, USA, 1:300), anti-Cyclin D1 (SP4, Thermo Fisher Scientific, Rockford, USA, 1:5) and anti-TP53 (clone DO-7, Dako, 1:3000). For staining quality checks positive control slides were included.

Scoring of immunohistochemical staining

All IHC results were examined independently by two observers (JHM and IG), who were blinded to the patients’ clinical information, using a high-power (400×) magnification. The labeling indices were determined by including all carcinoma cells within the tissue cores. For Reptin and Pontin nuclear and cytoplasmic, for β-catenin membranous, nuclear and cytoplasmic, and for TP53 only nuclear staining intensities were evaluated semi-quantitatively using the scores 0, 1, 2 or 3 through comparing the different tumor samples. For TP53 a sum score was calculated by multiplying the labeling index with the staining intensity. For MIB-1 and Cyclin D1 only the nuclear labeling indices were evaluated. Samples with discordant assessment results were re-evaluated till a consensus was reached.

Statistical analyses

All statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) for Mac OS X (Apple Inc., Cupertino, CA, USA). Associations between experimental findings and clinicopathological variables were assessed using Student’s t-test. Labeling indices were assessed as representing marker expression levels for all statistical evaluations. Correlations between marker expression levels were assessed using Spearman’s rank test. For survival analyses grouping was carried out relative to the mean labeling indices as cut-off points. Grouping according to the median labeling indices as cut-off points did not lead to any different results. For the different histopathological salivary gland tumor subtypes, marker profiles were defined according to their mean expression levels. The Kruskal-Wallis test was performed to detect significant differences between mean expression levels in SGC subtypes. OS rates were estimated using the Kaplan-Meier algorithm for incomplete observations and compared using the log-rank test. A p-value ≤ 0.05 was considered to be statistically significant.

Results and discussion

Despite significant progress that has been made in the prevention and management of SGC, the prognosis for patients with advanced stages of the disease is still poor. As yet, the therapeutic options beyond local therapy (surgery, adjuvant radiotherapy) are limited and little is known about the oncogenesis of this heterogeneous group of malignant tumors. Thus, currently the major goals in salivary gland cancer research include the generation of novel (targeted) therapies and unveiling the key molecular mechanisms driving tumor progression and metastasis. Here, we sought to determine the clinical and functional significance of expression of the AAA+ ATPases Reptin and Pontin in SGC.

The 94 SGC samples included in this study comprised 13 different histological subtypes (listed in Table 1). An overview of the various clinicopathological data is provided in Table 2. Using immunohistochemistry (IHC) we found that Reptin and Pontin were expressed in the majority of the SGC samples across all histological subtypes (Fig. 1a, b). The different SGC tumor cells displayed nuclear as well as cytoplasmic staining patterns. The percentages of positive cells and the staining intensities were variable for both IHC markers, with immune-positive tumor cells ranging from 0 to 100%. In Fig. 2 representative IHC staining patterns for the two markers are depicted. After analysis of the expression levels of these markers in relation to patient characteristics we found neither significant associations between Reptin or Pontin expression levels and the clinical or pathological staging variables T and N, nor between the subcellular localization (nuclear/cytoplasmic) of Reptin or Pontin and the occurrence of nodal or distant metastases. Reptin and Pontin are known to be frequently co-regulated and to occur in common protein complexes. Accordingly, we found in our 94 SGC samples that the sum scores of the nuclear staining intensities multiplied by the percentage labeling indices of Reptin and Pontin exhibited a weak correlation (r = 0.573; p = 0.01). In addition, we found that a low nuclear staining intensity of Pontin strongly correlated with both a low nuclear (p = 0.00001) and a low cytoplasmic (p < 0.0001) staining intensity of Reptin, as well as with a low percentage labeling index of Reptin (p = 0.003).

Fig. 1.

Distribution of immunohistochemical expression of Reptin (a) and Pontin (b) among the different histological salivary gland cancer tumor subtypes

Fig. 2.

Representative H&E and immunohistochemical stainings of the indicated markers in two tumors. First row: large cell undifferentiated carcinoma with strong Reptin and Pontin expression; second row: mucoepidermoid carcinoma with low Reptin and Pontin expression (original magnification 200×)

Reptin and Pontin have been reported to be abundantly expressed in tumor cells of various malignancies, whereas their expression in normal human tissues is tightly regulated [11, 15]. Functional studies have indicated that Reptin and Pontin are involved in multiple cellular processes including transcription regulation, chromatin remodeling and DNA damage repair [11, 15]. These processes are also frequently disrupted in cancer cells. In fact, Reptin has been implicated in the proliferation, invasion and metastasis of hepatocellular carcinoma and prostate cancer cells [30, 31]. Moreover, cytoplasmic expression of Pontin in renal cell carcinoma has been found to correlate with tumor cell invasion, metastasis and patient survival [32]. Thus, pathways regulated by Reptin and Pontin may be employed for the design of future therapeutic strategies, either by using specific inhibitors targeting Reptin or Pontin as distinct targets or by targeting protein complexes driven by these two AAA+ ATPases [11]. Interestingly, we found that a high percentage labeling index of Reptin (cut-off mean 79.66%) strongly correlated with a high expression of MIB-1 (p = 0.003) (Fig. 2), a proliferation marker that has been associated with a poor outcome in SGC patients [4, 33] and an increased risk of metastasis in various cancer types. IHC also revealed a significant correlation between Reptin expression (percentage labeling index, cut-off mean 79.66%) and TP53 accumulation (p = 0.042) as a surrogate mutant p53 marker (Fig. 2). In addition, we found that a high nuclear staining intensity of Reptin significantly correlated with a high expression of the cell cycle regulator Cyclin D1 (p = 0.006) (Fig. 2), which is often deregulated in human malignancies. Based on these observations, we assume that Reptin may also contribute to SGC progression and metastasis. No correlations were found between the Pontin and MIB-1, Cyclin D1 or TP53 expression levels in our SGC cohort. We also found that a strong nuclear staining of Reptin significantly correlated with cytoplasmic (p = 0.002), but not with nuclear (p = 0.135) expression of β-catenin. Only two cases with a high nuclear Reptin expression showed a concomitant high nuclear β-catenin expression. Nuclear accumulation of β-catenin is a prerequisite for its oncogenic function. Unexpectedly, we found that high nuclear Reptin levels did not correlate with nuclear β-catenin expression in most cases. It has been reported, however, that minor amounts of β-catenin may already be signaling competent and sufficient to induce the expression of target genes [34, 35]. In contrast, we found that nuclear Pontin expression significantly correlated with both cytoplasmic and nuclear β-catenin expression (p = 0.037 and p = 0.018, respectively). These observations suggest that in SGC Reptin and Pontin may act independently or even antagonistically, as has also been shown for regulation of the metastasis suppressor KAI1 [30] or Pontin, Reptin and β-catenin [23, 24, 36], respectively.

Next, we set out to assess the putative prognostic roles of Reptin and Pontin in SGC. We found that the five-year and ten-year overall survival (OS) rates in our patient cohort were 59.3 and 41.8%, respectively. We also found that patients with a high percentage labeling index of Reptin (cut-off mean 79.66%) showed a significant inferior five-year OS compared to patients with a low percentage labeling index of Reptin (47.7% versus 78.3%, respectively; p = 0.033) (Fig. 3a). The five-year OS of patients with a high percentage labeling index of Pontin (cut-off mean 54.32%) was not significantly different compared to patients with a low percentage labeling index of Pontin (57.8% versus 56.8%, respectively; p = 0.632) (Fig. 3b). For both nodal-negative patients and patients without adjuvant radiation therapy, the OS showed a strong trend in favor of patients with a low Reptin expression (p = 0.06 and p = 0.064, respectively) (Fig. 3c, d). However, due to the small number of patients in these subgroups this trend did not reach statistical significance. Our observation that patients with a high Reptin expression among patients without adjuvant radiation therapy, or nodal-negative patients, showed a strong trend towards inferior outcome may be indicative for a possible benefit of more aggressive therapeutic regimens in these patients. Future prospective studies are warranted to validate whether patients with early tumor stages and a concomitant high Reptin expression level should be considered for more radical surgical procedures and/or extended adjuvant therapy regimens.

Fig. 3.

Five-year overall survival (OS) according to Reptin or Pontin percentage labeling indices for all patients of the cohort (a and b) as well as for nodal-negative patients (c) and patients without adjuvant radiotherapy (d)

Here, we show for the first time that the AAA+ ATPases Reptin and Pontin are widely expressed in SGC tumor tissues and that high Reptin expression levels correlate with a dismal clinical outcome. Even though our study is limited due to the relatively small numbers of cases included in the different diagnostic subgroups, with 94 patients we still present a comparatively large SGC cohort, considering the low overall incidence of these tumors. Regarding the distribution of histological subtypes, age and sex, our cohort is in line with previous SGC studies. We conclude that Reptin and Pontin serve as novel markers that are frequently expressed in SGC. The observed correlation between a high Reptin expression, an increased MIB-1 expression and a decreased overall survival suggests a role of Reptin in SGC tumor progression and metastasis. Our results may pave the way for the design of novel diagnostic and (targeted) therapeutic strategies for this heterogeneous group of malignancies.

Funding

The W.E.B. laboratory is supported by the Deutsche Forschungsgemeinschaft (DFG), grant DFG EXC 1003, Cells in Motion, Cluster of Excellence.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.