Case Report
Transcriptional Levels of PRUNE-1 are Correlated to Prognostic Parameters in Epithelial Tumors
Larissa Vargas1, Sara TS Mota1, Sarah BR Nunes1, Fabrícia M Oliveira2, Galber R Araujo3, Adriana F Neves4, Yara CP Maia5, Luiz Ricardo Goulart2 and Thaise G Araújo1*
1Department of Biochemistry, Federal University of Uberlândia, Brazil
2Department of Mathematics, Federal University of Uberlândia, Brazil
3Department of Genetics and Biochemistry, Federal University of Uberlândia, Brazil
4Department of Laboratory of Molecular Biology, Federal University of Goiás GO, Brazil
5Department of Medicine, Federal University of Uberlândia, Brazil
*Corresponding author: Thaise G Araújo, Department of Biochemistry, Federal University of Uberlândia, Brazil
Published: 17 Aug, 2017
Cite this article as: Vargas L, Mota STS, Nunes SBR,
Oliveira FM, Araujo GR, Neves
AF, et al. Transcriptional Levels of
PRUNE-1 are Correlated to Prognostic
Parameters in Epithelial Tumors. Clin
Oncol. 2017; 2: 1327.
Abstract
Background: The human orthologue of Drosophila PRUNE protein (PRUNE-1) regulates cell
motility through its phosphodiesterase activity and interaction with protein partners, acting as a
negative regulator of Nm23-H1, a tumor suppressor protein. Here, we aimed to understand the
molecular alterations in epithelial tumors through quantification of PRUNE-1 transcriptional levels
in patients with Prostate Cancer (PCa) and Breast Cancer (BC).
Methods: mRNA expression was detected by RT-qPCR in peripheral blood from 31 PCa, 30 Benign
Prostatic Hyperplasia BPH, 48 Breast Cancer, and 41 Benign Breast Disease patients.
Results: Transcriptional levels of h-PRUNE in PCa were associated to high grade of Gleason
score. Regression analyses had shown correlation between Prostate Specific Antigen, Gleason and
PRUNE-1 mRNA expression. In BC, transcripts were significantly higher in HER-2 overexpression
group compared to luminal patients.
Conclusion: Up-regulation profile of PRUNE-1 may indicate convergent pathways for progression
and aggressive PCa and BC. Our results pointed PRUNE-1 as a putative gene for identify metastasis.
Keywords: Epithelial tumors; PRUNE-1; Transcription; Metastasis
Introduction
The PRUNE-1 (or h-PRUNE/DRES17) is the human homologue of the Drosophilagene that
encodes a member of the phosphodiesteraseprotein family (PDE). The PDE activity catalyzes the
hydrolysis of adenosine and guanosine-3’-5’-cyclic monophosphate (cAMP and cGMP) to their
inactive forms 5’-monophosphates. These cyclic nucleotide second messengers play a central role in
signal transduction and physiological responses. Binding to Nm23-H1, a tumor suppressor protein,
PRUNE-1 acts as a negative regulator, and with GSK-3β cooperatively promotes the disassembly of
focal adhesions [1].
The search for new putative targets in Prostate Cancer (PCa) and Breast Cancer (BC), the
most common malignant tumors after non-melanoma skin cancers, will help elucidate neoplastic
biology, particularly metastatic phenotype. In this brief report, we quantified, thought qPCR, the
transcriptional levels of PRUNE-1 in peripheral blood from patients with PCa; benign prostatic
hyperplasia (BPH); BC and benign breast disease (BBD) to better define the clinical and pathological
roles of this gene in epithelial tumors progression.
Patients and Methods
This study was conducted at the Laboratory of Nanobiotechnology of the Federal University of
Uberlândia (UFU), approved by UFU Research Ethics Committee (005/2001 and 176/2008). Blood
samples from 150 patients were grouped into four classes: 31 PCa; 30 BPH; 48 BC and 41 BBD.
Informed consent was obtained from all participants.
The average age of patients was 64 years old (range 51-82 years) for PCa group and 68 years old
(range 50-82 years) for BPH group. According to TNM classification, 18 (70%) and 8 (30%) prostate
tumors were T1/T2 and T3/T4, respectively. For Gleason score 40% presented Gleason ≥7 and 60%
Gleson <7. The average levels of serum total PSA for PCa was 7.5
(range from 1.9 to 13.44) and for BPH patients was 11.95 (range 2.92
to 18.16).
Considering BC patients, the average age was 55 years old (range
31–89 years) and for BBD was 48 years old (range 18–79 years).
Patients with BC were classified according to histological grading
(Nottingham system) as grade I (GI) in 3 (9%), GII in 25 (73%), and
GIII in 6 (18%). For hormone receptors 34 (85%) were positive for
Estrogen Receptor (ER) and 31 (80%) for Progesterone Receptor
(PR). HER-2 status was considered as positive (score 3+), including
19 cases (51%) and negative (score 0–1+) in 18 BC patients (49%). In
conclusive scores 2+ were excluded from statistical analyses.
Total RNA was extracted using TrizolLs Reagent (Invitrogen)
following the manufacturer's recommendations. Reverse transcription
and amplification reactions to validate RNA quality were conducted
as described elsewhere [2]. QPCR reactions were prepared to a final
volume of 10 μL, which was composed by 2 μL of cDNA, 5.0 μl of Master
Mix SYBR® Green reagent (Applied Biosystems), and 5 pmol of primers.
All reactions were conducted in an ABI 7300 Real Time PCR Systems
(Applied Biosystems) and analyzed according to delta threshold cycle
value (ΔCt) method. Pairs of primers were designed for PRUNE-1
amplification: forward 5’-CAGCCCTTCTGCATGGAAC-3’,
and reverse 5’-TTTCTCTTGGGTAGGTCTGG-3’ and Beta-2-
microglobulin transcripts were used for normalization as previously
described [2]. Mann-Whitney and regression analyses were carried
out to verify differences and correlations between groups. T-student
test was performed to verify significance of regression test. Statistical
significance was considered when p <0.05 and carried out using
GraphPad Prism 5 (GraphPad Soft-ware, La Jolla, CA, USA) and
Bioestat software.
Figure 1
Figure 1
Transcriptional quantification of PRUNE-1 in epithelial tumors: Prostate Cancer (PCa) and Breast Cancer (BC). (a) No difference between PCa and
Benign Prostatic Hyperplasia (BPH)samples (b)The transcripts were significantly different in patients with Gleason score ≥7. *p=0.029 (c) No difference between
BC and Benign Breast Disease (BBD) samples (d) Higher levels of PRUNE-1 were detected in HER-2 over expression group **p=0.048. TN: triple-negative breast
cancer.
Results
The distribution of PRUNE-1 mRNA in the peripheral blood was
not different among PCa and BPH groups. Patients with Gleason
≥7 presented1.7 fold higher transcriptional (Figure 1a and b) (p =
0.029) and it was not found any significant association to other
clinicopathological features. Positive correlation between Gleason
(X axis) and PRUNE-1 (Y axis) mRNA expression was verified
through univariate regression (y = 6.0610 + 0.5754x and R2 = 0.0772).
Multivariate regression demonstrated a negative correlation to
PSA(X1) (y = - 0.3773 - 0.0321 X1 + 0.2173 X2 and R2 = 0.0815).
In BC, there were no significant difference in PRUNE-1 expression
between tumor and benign samples. Transcripts amplified from
peripheral blood did not also demonstrate significant differences
according to clinicopathological data. Considering intrinsic subtypes,
transcriptional levels were significantly associated to HER-2 over
expression group (p = 0.048) comparing to luminal tumors (Figure
1c and d).
Discussion
Over expression of PRUNE-1 in breast, colorectal, and gastric
cancers is correlated with lymph node and distant metastases, once
increased levels of this gene may function as a negative regulator of
tumor suppressor activities of nm23-H1. In fact, amplification of
1q21-q22 had already been described as a common molecular event
in metastatic lesions associated with poor outcome [3].
The survival of patients with PCa is related to several factors,
including extent of tumor, histologic grade, patient’s age, and PSA
levels. In accordance with Gleason system, prostate cancers with high
grade (poorly differentiated) tend to be aggressive and they are likely
to grow and spread quicker. Better prognostic is related to lower
score and this is one of the most important prognostic factors for
PCa treatment [4]. We found that prostate cancer showed increased
levels of h-PRUNE in higher Gleason score, suggesting that it plays an
important role in cancer development, thus serving as a progression
marker.
Over expression of PRUNE-1 in BC has also been associated to
cell motility, lymph node involvement and metastasis formation
[5]. Comparing the expression levels of the mRNA marker with the
clinicopathological features we found an up-regulation in HER-
2 subtypes comparing to luminal one. The HER2-2 oncogene is
amplified and/or overexpressed in approximately 20% of breast
cancers. This marker predicts for trastuzumab adjuvant therapy and
is a strong prognostic factor for relapse and poor overall survival [6].
Therefore, the correlation between HER-2 subtype and PRUNE-1
up-regulation may indicate convergent pathways for progression
and metastatic BC. Our work demonstrated an association between
high levels of h-PRUNE RNA and aggressiveness of BC and PCa. The
identification of molecules that are capable of in hibith – PRUNE
signaling pathway are of great relevance for the development of new
drugs to treat cancer metastasis. Further studies with larger cohorts
including patients undergoing different outcomes are truly necessary
to better understand the real clinical relevance of h-PRUNE pathway.
References
- Tammenkoski M, Koivula K, Cusanelli E, Zollo M, Steegborn C, Baykov AA, et al. Human Metastasis Regulator Protein H-Prune is a Short-Chain Exopolyphosphatase. Biochemistry. 2008; 47(36): 9707-9713.
- Araujo TG, Marangoni K, Rocha RM, Maia YC, Araujo GR, Alcântar TM, et al. Dynamic dialog between cytokeratin 18 and annexin A1 in breast cancer: A transcriptional disequilibrium. Acta Histochemica. 2014; 116(7): 1178-1184.
- Forus A, D'Angelo A, Henriksen J, Merla G, Maelandsmo GM, Flørenes VA, et al. Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas-a possible mechanism for altering the nm23-H1 activity. Oncogene. 2001; 20(47): 6881-6890.
- Epstein JI. An update of the Gleason grading system. J urol. 2010; 183(2): 433-440.
- Zollo M, Andrè A, Cossu A. Overexpression of h-prune in Breast Cancer is Correlated with Advanced Disease Status. American Association for Cancer Research. 2005; 11(7): 199-205.
- Huszno J and Nowara E. Risk factors for disease progression in HER2-positive breast cancer patients based on the location of metastases. Przeglad menopauzalny = Menopause review. 2015; 14(3): 173-177.