Autophagy deactivation is associated with severe prostatic inflammation in patients with lower urinary tract symptoms and benign prostatic hyperplasia

Abstract

Autophagy is a conserved evolutionary process that allows cells to maintain macromolecular synthesis and energy homeostasis during starvation and stressful conditions. We prospectively evaluated the relationship between autophagy and prostatic inflammation in a series of transurethral prostatic resection samples. Inflammatory infiltrates were defined according to the standardized classification of chronic prostatitis of the National Institute of Health. The inflammatory score (IS score) was calculated. High IS score was defined as ≥7. Each sample was stained for anti-LC3B and for anti-P62/SQSTM1 and scored. High p62 or LC3B percentage was defined as >25%, whereas low was defined as <25% of cells with dots.
We analyzed 94 specimens. Overall, 18/94 (19%) showed no sign of prostatic inflammation, whereas 76/94 (81%) presented inflammatory infiltrates. Inflammation was mild in 61/76 (80%), moderate/severe in 15/76 (20%). Patients with high p62 percentage were 62/94 (66%) while 32 (34%) showed low p62 percentage. Patients with high LC3B percentage were 37/94 (39%) while 57(61%) showed low LC3B percentage. Overall 42/94 (44%) patients presented a high p62 percentage and concomitant a low LC3B percentage. IS score was significantly higher in patients with a with high p62 percentage (median IS 7 (6/8) vs 5 (3/7); p= 0.04) and in patients with a low LC3B percentage (median IS 7 (6/8) vs 5 (3/7); p= 0.004) when compared to patients with a low p62 percentage or a high LC3B percentage respectively. On multivariate analysis, p62 (OR: 10.1, 95%CI: 2.6-38.6; p= 0,001) and LC3B expression (OR: 0.319; 95%CI: 0.112-0.907; p= 0.032) were independent predictors of a high IS.
Here we present the first evidence of autophagy deregulation in prostatic inflammation. These results raise many questions about the mechanisms mediating the autophagy dysfunction and the links to prostatic inflammation that need to be addressed.

Full text

Oncotarget1
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Autophagy deactivation is associated with severe prostatic
inammation in patients with lower urinary tract symptoms and
benign prostatic hyperplasia
Cosimo De Nunzio1, Simona Giglio2, Antonella Stoppacciaro2, Mauro Gacci4,
Roberto Cirombella2, Emidio Luciani2, Andrea Tubaro1, Andrea Vecchione2,3
1Urology Units, Department of Clinical and Molecular Medicine, Ospedale Sant’Andrea, Sapienza University, Rome, Italy
2Surgical Pathology Units, Department of Clinical and Molecular Medicine, Ospedale Sant’Andrea, Sapienza University, Rome,
Italy
3Department of Cancer Biology and Genetics/CCC, The Ohio State University, Columbus, OH, USA
4Department of Urology, Careggi Hospital, Firenze, Italy
Correspondence to: Cosimo De Nunzio, email: cosimodenunzio@virgilio.it
Keywords: autophagy, BPH, LUTS, inammation, prostate
Received: December 02, 2016 Accepted: January 11, 2017 Published: February 07, 2017
ABSTRACT
Autophagy is a conserved evolutionary process that allows cells to maintain
macromolecular synthesis and energy homeostasis during starvation and stressful
conditions. We prospectively evaluated the relationship between autophagy and
prostatic inammation in a series of transurethral prostatic resection samples.
Inammatory inltrates were dened according to the standardized classication
of chronic prostatitis of the National Institute of Health. The inammatory score (IS
score) was calculated. High IS score was dened as ≥7. Each sample was stained for
anti-LC3B and for anti-P62/SQSTM1 and scored. High p62 or LC3B percentage was
dened as >25%, whereas low was dened as <25% of cells with dots.
We analyzed 94 specimens. Overall, 18/94 (19%) showed no sign of
prostatic inammation, whereas 76/94 (81%) presented inammatory inltrates.
Inammation was mild in 61/76 (80%), moderate/severe in 15/76 (20%). Patients
with high p62 percentage were 62/94 (66%) while 32 (34%) showed low p62
percentage. Patients with high LC3B percentage were 37/94 (39%) while 57(61%)
showed low LC3B percentage. Overall 42/94 (44%) patients presented a high p62
percentage and concomitant a low LC3B percentage. IS score was signicantly
higher in patients with a with high p62 percentage (median IS 7 (6/8) vs 5 (3/7);
p= 0.04) and in patients with a low LC3B percentage (median IS 7 (6/8) vs 5 (3/7);
p= 0.004) when compared to patients with a low p62 percentage or a high LC3B
percentage respectively. On multivariate analysis, p62 (OR: 10.1, 95%CI: 2.6-38.6;
p= 0,001) and LC3B expression (OR: 0.319; 95%CI: 0.112-0.907; p= 0.032) were
independent predictors of a high IS.
Here we present the rst evidence of autophagy deregulation in prostatic
inammation. These results raise many questions about the mechanisms mediating
the autophagy dysfunction and the links to prostatic inammation that need to be
addressed.
INTRODUCTION
Lower urinary tract symptoms (LUTS) and Benign
Prostatic Hyperplasia (BPH) are highly prevalent in adult
males and BPH is the most frequent benign neoplasm
in aging men [1]. Although several mechanisms seem
to be involved in its development and progression the
pathogenesis of this condition is still largely unknown. In
www.impactjournals.com/oncotarget/ Oncotarget, Advance Publications 2017

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the past few years recent evidence clearly suggested the
possible role of prostate inammation in the pathogenesis
of LUTS and BPH [2–5].
Autophagy is a highly conserved evolutionary
process that is involved in a number of cellular
homeostatic processes that allows the cell to maintain
macromolecular synthesis and energy homeostasis
during starvation and other stressful conditions [6, 7].
Therefore induction of autophagy exerts anti-aging and
oncosuppressive functions. A hallmark of autophagy
is the formation of the autophagosome (double-
membrane cytosolic vacuoles), in which proteins and
organelles are imbibed, and then after fusion with
lysosomes are degraded. Autophagy is regulated by
a series of related genes. In particular two ubiquitin
like conjugation system (ATG12-ATG5-ATG16 and
ATG8) are crucial for autophagosome formation and
cargo recruitment. One of the human homologue of
ATG8 (LC3B) represents one of the most widely used
markers to monitor this process [8]. Autophagy can be
a highly selective process, which is achieved through
receptors that are able to interact with the autophagy
machinery and to recognize ligand bound cargo.
One the best studied receptor is p62, also known as
sequestosome (SQSTM1). Since p62 is localized to the
autophagosome via LC3-interaction and is constantly
degraded by the autophagy–lysosome system, therefore
autophagy inhibition leads to the accumulation of p62
positive aggregates [9]. Recent studies [7, 10–12] have
shown that in adipocytes or in pancreatic cells the levels
of inammatory gene and cells, and the activation
of several inammatory pathways are inuenced by
autophagy activation/deactivation. Furthermore there
is an intense investigation on targeting autophagy
mechanism in several malignancies including prostate
cancer. With this knowledge in mind we hypothesized
that autophagy could play a role in the prostate cells
immune response with a subsequent effect on prostatic
inammation. To this aim we evaluated the relationship
between autophagy and prostatic inammation assessing
the expression of autophagy markers P62 and LC3B.
RESULTS
We analyzed 94 surgical specimens of TransUrethral
Resection of Prostate (TURP). Patients’ characteristics
are summarized in Table 1. Overall, 18/94 (19%)
showed no sign of prostate inammation at the histology
report, whereas 76/94 subjects (81%) presented an
inammatory inltrate. Inammation was mild in 61/76
(80%) and moderate/severe in 15/76 (20%). The median
inammatory score was 6 (IQR: 4-7). Overall 60 patients
(64%) presented a low inammatory score (IS) (IS < 7)
and 34 (36%) a high inammatory score (IS ≥ 7).
Patients with prostate inammation (IS ≥ 7)
presented an higher pre-operative International Prostatic
Symptom Score (IPSS), when compared to those without
an IS < 7 (Table 1).
Patients with high percentage of p62 were 62/94
(66%) while patients with low percentage were 32/94
(34%). Patients with high percentage of LC3B were
37/94 (39%) while patients with low percentage of were
57/94 (61%). Overall 42/94 (44%) patients presented
a low percentage of LC3B and a concomitant high
percentage of p62 (high p62/low LC3B). Patients with an
IS ≥ 7 presented a higher percentage of p62 and a lower
percentage of LC3B when compared to patients with a
lower IS score (Table 2).
IS score was signicantly higher in patients
with high percentage of p62 [median IS: 7 (6/8) vs 5
(3/7); p= 0.04] and in patients with a low percentage of
LC3B [median IS: 7 (6/8) vs 5 (3/7); p= 0.004] when
compared to patients with a low percentage of p62 and
a high percentage of LC3B respectively. On multivariate
analysis, p62 (OR: 10.1, 95%CI: 2.6-38.6; p= 0,001)
and LC3B expression (OR: 0.319; 95%CI: 0.112-0.907;
p= 0.03) were independent predictors of a high IS. Age
was not associated to an increased risk of inammatory
inltrates (OR: 1.02; 95%CI: 0.956-1.103; p= 0.46).
DISCUSSION
The presence of chronic histological inammation
is a well-known nding in biopsy and surgical specimens
of prostate tissue in patients with and without lower
urinary tract symptoms or prostatitis [3]. Histological
inammation was found in more than 78% of men
enrolled in the Reduction by Dutasteride of Prostate
Cancer Events trial (REDUCE), demonstrating its
ubiquitous nature in aging men, although its relation to
histological and clinical BPH is unclear [13]. In our study,
most of our patients (81%) with LUTS/BPH treated with a
TURP, presented an inammatory inltrate. Inammation
was moderate/severe in about 20% of the study
population. As recently proposed in several studies [5, 14]
investigating the relationship between inammation and
LUTS, prostatic inammatory inltrates were dened
according to the standardized classication system of
chronic prostatitis (CP-CPPS) of the National Institutes of
Health (NIH), including the inammatory score [15]. We
conrmed as in previous experiences [16] that a higher IS
was observed in about 40% of the study population and
it was associated with a more severe IPSS. Patients with
prostate inammation (IS ≥ 7) presented an higher pre-
operative IPSS, and particularly an higher IPSS storage
subscore. Our data also conrmed a previous experience
where metabolic syndrome associated with inammatory
inltrates through different mostly unknown mechanism
signicantly increases the risk of an IPSS storage
subscore ≥ 4 (OR: 1.782; 95%CI 1.045-3.042; p = 0.030)
[17, 18].

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Autophagy is a key process for the regular
maintenance and disposal of intracellular organelles
and proteins [7, 19]. As observed in other tissues we
hypothesized that autophagy could play an important role
in regulating the maintenance of accumulated molecules
with a subsequent effect on prostatic inammation [7].
In our study we showed that autophagy is suppressed
in the prostatic cells in the presence of a signicant
prostatic inammatory inltrates (IS > 7). Indeed in
this group of patients we observed high expression of
p62 and a low level of LC3B. Particularly the positive
expression of p62 increased by ten times the risk of
severe prostatic inammation raising the question of the
potential role of autophagy in inammatory response in
patients with LUTS/BPH. Thus inhibition of autophagy
through different unknown mechanisms may activate the
inammatory response in the prostate by the increased
expression of pro-inammatory genes/cytokines and
decreased expression of anti-inammatory genes/
cytokines as observed in the adipocytes or in pancreatic
cells [7, 19]. Particularly, evidence from animal models
indicates that autophagy is impaired in pancreatitis, and
that one possible mechanism involved is the defective
functions of lysosomes. As for prostatic inammation in
our study, pancreatitis decreases autophagy efciency by
an increased level of p62, a multifunctional protein that
mediates autophagic clearance of ubiquitinated protein
aggregate [11]. The p62 accumulation in autophagy-
decient cells also leads to NF-kb activation, clears
apoptotic material which induces tissue inammation
or could increase levels of ROS which is required for
inammasone activation, a complex of cytosolic proteins,
secerned by immune cells (macrophages and dendritic
cells) in response to different “danger signals” which
cleaves pro-IL-18 to the mature form and further increases
its secretion from immune cells. Inammasome, ROS
and IL-8 activities have been recently associated to the
activation of the prostatic associated lymphoid tissue
and the development of prostatic inammatory inltrates
[20–22] with a subsequent inammatory tissue damages
and continuative wound healing nally may induce the
development of BPH nodules. Recent data also suggested
a possible role of autophagy dysregulation in prostate
cancer development and progression. Burdelski C et al.
[23] in a immunohistochemistry tissue microarray study
of 12,427 prostate cancers, demonstrated that strong
Table 2: Patient’s characteristics according to the autophagy protein expression and inammatory score
Overall Inammatory score <7 Inammatory score ≥7 p
High% p62 62/94 (66%) 29/60 (48%) 31/34 (91%) 0.001
Low% LCB3 57/94 (60%) 31/60 (51%) 26/34 (76%) 0.015
High p62/Low BC 42/94 (45%) 18/60 (30%) 24/34 (70%) 0.001
Table 1: Patient’s characteristics according to the presence of prostatic inammation
Overall Inammatory score < 7 Inammatory score ≥ 7 p
Patients 94 60/94 (64%) 34/94 (36%)
Age (years) 69,6 ± 6.8 (69; 65-75) 69.6±7.1 (69; 66-75) 69.6±6.5 (71; 65-74.5) 0.779
BMI (kg/m²) 22,9±2.8 (22.4; 21-24) 22.7±2.9 (22.6; 20-24) 23.1±2.8 (22.4; 21-24) 0.484
PSA (ng/ml) 6±4 (5.2; 2.7-9.1) 6.5±5.2 (5; 2-9.2) 6±4 (4.7; 3.3-9) 0.865
TRUS Volume (ml) 71 ±17 (66; 50-96) 70±28.1 (65; 50-89) 65.8±22.5 (59.5; 50-82) 0.247
IPSS 18.9 ±6.7 (18; 13-24) 17.4±6.1 (16; 13-23) 21.4±7 (24; 16-2721) 0.004
IPSS voiding 9.3±3.7 (10; 6-12) 8.9±3.5 (8; 6-12) 10.1±3.9 (11; 6-12) 0.123
IPSS storage 9 ± 4 (9.5; 6-12) 8.6±4.1 (7.5; (6-12) 10.5 ± 3.9 (11; 9-13) 0.018
Qmax (ml/s) 8.7±2.8 (8.2; 6.1-10.6) 6±1 (6; 4-6) 8.7±2.9 (9; 6-10) 0.756
PVR (ml) 44.6±38.4 (37.5; 17.5-69.2) 33±15 (30; 20-50) 43.7±49.4 (30; 0-88) 0.421
Data are presented as mean ± DS (median; IQR); BMI: Body Mass Index; PSA: Prostate Specic Antigen; TRUS: Trans
Rectal UltraSound; IPSS: International Prostate Symptoms Score; Qmax: maximum urine ow; PVR: Post Voiding
Residual.

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cytoplasmic p62 staining was linked to high Gleason
grade, advanced pathologic tumor stage and early PSA
recurrence. Analysis of cytoplasmic accumulation of p62
was considered a strong predictor of an adverse prognostic
behavior of prostate cancer. Considering the possible link
between inammation and prostate cancer development
and progression [3] the role of autophagy could be
twofold.
Although what we know is only the tip of a
very large iceberg and the evidence on the molecular
mechanism behind the relationship between autophagy
and prostatic inammation is quite primitive, elucidating
these possible mechanisms could lead to the identication
of new therapeutic targets acting to normalize the
autophagy function.
We must acknowledge some limitations to our
study: it is a single center study with a small number of
patients. Furthermore, no specic serum or molecular
markers of prostatic inammation were used and the
authophagy status was evaluating exclusively using
the expression of autophagy markers p62 and LC3B.
Although, inammatory markers is an interesting topic
as they could be used to better identify patients with
prostatic inammation, at this stage the question on what
is the gold standard marker for prostatic inammation
continues to be debated and as a consequence no specic
prostatic inammatory markers are routinely available
in our clinic.
Although detection of autophagosomes by electron
microscopy is still regarded as the gold standard to
detect autophagy in tissue, this method is time and cost
consuming and restricted to the application on non
formalin xed and parafn embedded tissue. Therefore
we use two of the major autophagy proteins, which have
been validated in different studies [6, 24].
Our results apply to this study (patients with
BPH and LUTS resistant to medical therapy or with
chronic urinary retention treated with TURP) and
cannot be extended to all patients at risk for LUTS.
Notwithstanding all these limitations, it is the rst
study investigating the relationship between the key
structural authophagosomal proteins p62 and LC3B,
involved in delivery of damaged proteins mitochondria
to authophagosomes, and inammatory inltrates in
patients with LUTS/BPH treated with TURP. The
current pilot study suggests that autophagy is an
important process in prostatic inammatory inltrates
development and progression, and could be considered
a new possible target for the management of prostatic
disease. Immunohistochemical assessment of key
autophagy proteins, such as p62 and LC3B, is feasible
and their expression may identify a group of patients
with severe prostatic inammatory inltrates. These
ndings should be conrmed by further larger series
of patients with prostatic diseases and further studies
should also evaluate deeper insight the possible link
between autophagy defect and prostatic inammation
associated metabolic diseases such as obesity and
metabolic syndrome as recently observed for pancreatic
disorders [11].
Figure 1: Staining of LC3B and P62 in prostate glands in different inammatory conditions. A-C. A, Inammation score
8 prostatitis (A, 20x) showing LC3B negative staining (B, 40x) and P62 dot-like positive staining score 3 (C, D 40x). D-F. Inammation
score 8 prostatitis (A, 20x) LC3B dot-like positive staining +2 score (E, 40x) and P62 negative staining (F, 40X) are shown.

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MATERIALS AND METHODS
Patients
From April 2014 to September 2015, a consecutive
series of patients treated in our center with monopolar
TURP were prospectively included in this study.
Indications for surgery were LUTS/BPH resistant to
medical treatment and chronic urinary retention. Our
Ethical Committee approved the study and all patients
signed a dedicated informed consent. Exclusion
criteria included history of bladder or prostate cancer,
chronic prostatitis, bladder stones, urethral stenosis and
neurological diseases. Age, co-morbidities, anthropometric
parameters including body mass index (BMI) were
recorded for all patients. At the baseline all men were
evaluated with the IPSS, an uroowmetry was also
recorded. Additionally, prostate volume was evaluated
by means of trans-rectal ultrasound. A series of TURP
samples were included in this study and evaluated for the
presence of prostatic inammatory inltrates and for the
expression of the autophagy proteins p62 and LC3B.
Prostatic inammatory assessment
According to the standardized classication system
of chronic prostatitis (CP-CPPS) proposed by Nickel et
al in 2001 [15], all TURP specimens were examined to
dene the grade (no inammatory cells, mild inammation
with scattered inammatory cells, moderate inammation
characterized by non-conuent lymphoid nodules, severe
inammation dened by large areas of conuent inltrates)
the anatomical location (glandular, periglandular and/
or stromal), and the extent (focal <10%, multifocal 10-
50%, diffuse >50%) of the prostatitis. The inammatory
score (IS score) was calculated as the sum of the three
different histological inammatory parameters (anatomical
location, grade, and extent), each parameter ranges from 1
to 3. High IS score was dened as ≥ 7 [15].
Immunohistochemical procedure and evaluation
Immunohistochemistry was performed as previously
described [23]. Briey, after deparafnization, all sections
were immunostained with a 1:200 dilution of the anti-
LC3B (Cell Signaling, Lausen, Switzerland) and 1:200 of
the anti-anti-p62/SQSTM1 antibodies (MBL, Nunningen,
Switzerland). The primary antibody was omitted and
replaced with preimmune serum in the negative control.
Sections were reacted with biotinylated anti-rabbit
antibody and streptavidin-biotin-peroxidase (Dako
Laboratories, San Francisco, CA). Diaminobenzidine was
used as a chromogene substrate. Finally, sections were
washed in distilled water and weakly counterstained with
Harry's modied hematoxylin. All sections were examined
independently by two investigators (A.V., A.S.). Any
positive reaction was scored as follow: 0 = No dots; 1 =
detectable dots in 5-25% of cells; 2 = readily detectable
dots in 25-75% of cells; 3 = dots in >75% of cells. High
percentage of p62 or LC3B was dened as >25%, whereas
low percentage of p62 or LC3B was dened as <25% of
cells with dots.
The diffuse cytoplasmic reactivity of p62
antibodies and LC3B was assessed semi quantitatively.
The intensity of the cytoplasmic pattern was evaluated
as the proportion of cells with a strong, weak, or absent
reactivity in all available optical elds of a tissue section
at 20X magnication. The mean value was taken into
consideration. Cases without any expression were
considered as being negative. Thus, three distinct groups
were created: (1) negative/weak expression, (2) strong
expression in ≤50% of cells (limited over expression),
and (3) strong expression in >50% of cells (extensive
over expression). The percentage of cells with nuclear
p62 expression was also assessed in all optical elds
(magnication 20X). Cases with nuclear staining in >50%
of cells were considered as positive; the remaining were
recorded being negative (Figure 1).
Statistical analysis
Statistical analysis was performed using the S-PSS
12.0 software. Evaluation of data distribution showed a
non-normal distribution of the study data set. Differences
between groups of patients in medians for quantitative
variables and differences in distribution for categorical
variables were tested with the Kruskal Wallis one-way
analysis of variance and chi-square test, respectively.
We conducted an uni-multivariate logistic regressions
to assess the association between autophagy proteins
expression and the overall risk of prostatic inammation.
The variables considered for entry into the model were
age, p62 and LC3B (categorical variables). An alpha value
of 5% was considered as threshold for signicance. Data
is presented as median [Inter quartile range (IQR), mean ±
standard deviation (SD)].
CONCLUSIONS
Here we present the rst evidence of autophagy
deregulation in prostatic inammation. These results
raise many questions about the “upstream” mechanisms
mediating the autophagy dysfunction and the
“downstream” links to prostatic inammation that need to
be addressed. Answers to these questions will provide new
insight into molecular targets and therapeutic strategies for
treatment of prostatic diseases.
Abbreviations
LUTS: Lower urinary tract symptoms; BPH: Benign
Prostatic Hyperplasia; SQSTM1: Sequestosome; TURP:

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TransUrethral Resection of Prostate; IS: Inammatory
Score; REDUCE: Reduction by Dutasteride of Prostate
Cancer Events trial; CP-CPPS: Classication System of
Chronic Prostatitis; NIH: National Institutes of Health;
IPSS: International Prostate Symptoms Score; MTOPS:
Medical Therapies of Prostate Symptoms; BMI: Body
Mass Index; IQR: Inter Quartile Range; SD: Standard
Deviation
ACKNOWLEDGMENTS
Declared none.
CONFLICTS OF INTEREST
All authors declare that there are no conicts of
interest.
FUNDING
This work was partially supported by grant from
Associazione Italiana Ricerca sul Cancro (AIRC IG 16862
to AV).
Author contributions
All authors made a substantial contribution to
researching data for this article, discussions of content,
writing the article and editing and/or reviewing the
manuscript before submission.
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