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\begin{document}

\title{Mechanism of drug resistance to first-line chemotherapeutics mediated by
TXNDC17 in neuroblastomas}



\author[1]{Chenggong Zeng}%
\author[1]{Zhuoran Li}%
\author[1]{Zhiqing Wei}%
\author[1]{Tingting Chen}%
\author[1]{Juan Wang}%
\author[1]{Junting Huang}%
\author[1]{Feifei Sun}%
\author[1]{Jia Zhu}%
\author[1]{Suying Lu}%
\author[1]{Zijun Zhen}%
\affil[1]{Sun Yat-sen University State Key Laboratory of Oncology in South China}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
The prognosis of high-risk neuroblastomas (NB) that are resistant to
first-line induction chemotherapy is relatively poor. This study
explored the mechanism of resistance to first-line chemotherapeutics
mediated by TXNDC17 and its potential solutions in NB. The genetic and
clinical data of patients with NB were obtained from the Therapeutically
Applicable Research to Generate Effective Treatments dataset. TXNDC17
and BECN1 expressions in NB cells were up- and downregulated by
transfection with plasmids and shRNA, respectively. Autophagy-related
proteins were detected by western blot. Cell viability was determined
using cell proliferation and toxicity experiments. Apoptotic cells were
detected using flow cytometry. Overall, 1076 pediatric and adolescent
patients with NB were enrolled in this study. The 10-year overall
survival (OS) rates and event-free survival (EFS) rates for the patients
with mutation of BECN1 were 37.4\% \selectlanguage{ngerman}± 9.1\% and 34.5\% ± 8.8\%,
respectively. For patients with mutation of TXNDC17, the 10-year OS and
EFS were 41.4\% ± 5.9\% and 24.3\% ± 5.1\%, respectively, which were
significantly lower than those in the unaltered group. The
overexpression of BECN1 and TXNDC17 reduced NB sensitivity to cisplatin
(DDP), etoposide (VP16), and cyclophosphamide (CTX). Autophagy mediated
by BECN1 was regulated by TXNDC17, and this process was involved in the
resistance to DDP, VP16, and CTX in NB. Suberoylanilide hydroxamic acid
(SAHA) can enhance the sensitivity of NB cells to chemotherapeutics by
inhibiting TXNDC17, ultimately decreasing autophagy-mediated
chemoresistance. Acquired resistance to first-line chemotherapeutics was
associated with autophagy mediated by BECN1 and regulated by TXNDC17,
which can be reversed by SAHA.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Original Article}

\textbf{Title Page}

\textbf{Title:}

Mechanism of drug resistance to first-line chemotherapeutics mediated by
TXNDC17 in neuroblastomas

\textbf{Authors' names:}

Chenggong Zeng\textsuperscript{1,2,3,*}, Zhuoran
Li\textsuperscript{1,2,3,*}, Zhiqing Wei\textsuperscript{1,2,3,*},
Tingting Chen\textsuperscript{1,2,3}, Juan Wang\textsuperscript{1,2,3},
Junting Huang\textsuperscript{1,2,3}, Feifei Sun\textsuperscript{1,2,3},
Jia Zhu\textsuperscript{1,2,3}, Suying Lu\textsuperscript{1,2,3}, and
Zijun Zhen\textsuperscript{1,2,3}

*These authors contributed equally: Chenggong Zeng, Zhuoran Li, and
Zhiqing Wei

\textbf{Authors' affiliations:}

\textsuperscript{1}State Key Laboratory of Oncology in South China,
Guangdong Provincial Clinical Research Center for Cancer,Sun Yat-sen
University Cancer Center, Guangzhou 510060, P. R. China.

\textsuperscript{2}Collaborative Innovation Center of Cancer Medicine,
Sun Yat-sen University Cancer Center, Guangzhou 510060, P. R. China.

\textsuperscript{3}Department of Pediatric Oncology, Sun Yat-sen
University Cancer Center, Guangzhou 510060, P. R. China.

\textbf{Corresponding author:}

Zijun Zhen,\emph{zhenzj@sysucc.org.cn}

\textbf{Keywords:}

Neuroblastoma, drug-resistance, autophagy, BECN1, TXNDC17

\textbf{Abstract}

The prognosis of high-risk neuroblastomas (NB) that are resistant to
first-line induction chemotherapy is relatively poor. This study
explored the mechanism of resistance to first-line chemotherapeutics
mediated by TXNDC17 and its potential solutions in NB. The genetic and
clinical data of patients with NB were obtained from the Therapeutically
Applicable Research to Generate Effective Treatments dataset. TXNDC17
and BECN1 expressions in NB cells were up- and downregulated by
transfection with plasmids and shRNA, respectively. Autophagy-related
proteins were detected by western blot. Cell viability was determined
using cell proliferation and toxicity experiments. Apoptotic cells were
detected using flow cytometry. Overall, 1076 pediatric and adolescent
patients with NB were enrolled in this study. The 10-year overall
survival (OS) rates and event-free survival (EFS) rates for the patients
with mutation of BECN1 were 37.4\% \selectlanguage{ngerman}± 9.1\% and 34.5\% ± 8.8\%,
respectively. For patients with mutation of TXNDC17, the 10-year OS and
EFS were 41.4\% ± 5.9\% and 24.3\% ± 5.1\%, respectively, which were
significantly lower than those in the unaltered group. The
overexpression of BECN1 and TXNDC17 reduced NB sensitivity to cisplatin
(DDP), etoposide (VP16), and cyclophosphamide (CTX). Autophagy mediated
by BECN1 was regulated by TXNDC17, and this process was involved in the
resistance to DDP, VP16, and CTX in NB. Suberoylanilide hydroxamic acid
(SAHA) can enhance the sensitivity of NB cells to chemotherapeutics by
inhibiting TXNDC17, ultimately decreasing autophagy-mediated
chemoresistance. Acquired resistance to first-line chemotherapeutics was
associated with autophagy mediated by BECN1 and regulated by TXNDC17,
which can be reversed by SAHA.

\textbf{Abbreviations:}

NB, neuroblastoma; OS, overall survival; EFS, event-free survival; DDP,
cisplatin; VP16, etoposide; CTX, cyclophosphamide; SAHA, suberoylanilide
hydroxamic acid; CQ, chloroquine; HDACI, hydroxamic histone deacetylase
inhibitor; TRX, thioredoxin; TARGET, Therapeutically Applicable Research
to Generate Effective Treatments.

\textbf{Introduction}

Neuroblastoma (NB), the most common extracranial solid tumor in children
and adolescents, originates from primitive neural crest cells and
accounts for approximately 10\% of pediatric tumors\textsuperscript{1}.
By combining various clinical and molecular risk factors, patients with
NB can be stratified into low-, intermediate-, and high-risk groups.
Unfortunately, the long-term survival rates for patients with high-risk
NB are still approximately 40--60\% despite aggressive multimodal
treatment\textsuperscript{2-4}.

NB is a chemosensitive tumor, and chemotherapy plays an important role
in its treatment. Induction chemotherapy can effectively reduce the
tumor burden and create conditions for achieving local control.
Furthermore, tumor response to induction chemotherapy can guide
subsequent treatments\textsuperscript{5, 6}. The long-term survival
rates are \textless{}20\% for patients with high-risk NB who respond
poorly to induction chemotherapy\textsuperscript{6-8}. Currently, most
induction regimens for high-risk NB utilize a combination of
chemotherapeutics, including anthracyclines, platinum, alkylating
agents, and topoisomerase II inhibitors\textsuperscript{9, 10}. The
initial response rates are commonly high; however, drug resistance
develops in some patients as the treatment continues. However, the exact
underlying mechanism remains unclear.

Autophagy is a physiological process that occurs in response to
metabolic stress\textsuperscript{11}. Autophagy is one of the modalities
of cell death that occurs after chemotherapy and is also one of the
reasons for chemoresistance\textsuperscript{12-14}. Our previous study
confirmed that resistance to cisplatin (DDP), etoposide (VP16), and
cyclophosphamide (CTX) in NB cells was promoted by
autophagy\textsuperscript{15}. Chloroquine (CQ), the most commonly used
autophagy inhibitor, reduces the resistance of tumor cells to
chemotherapeutics by blocking the final stage of
autophagy\textsuperscript{15, 16}. However, CQ has some limitations in
clinical practice for cancer treatment\textsuperscript{17, 18}.

As a major autophagy regulator, BECN1 plays an important role in the
formation, elongation, and maturation of the
autophagosome\textsuperscript{19, 20} and can be directly regulated by
several cellular signals through posttranslational
modifications\textsuperscript{21, 22}. TXNDC17 is a new member of the
thioredoxin family with a molecular mass of 14 kDa, and was recently
found to be expressed in multiple types of human
tissues\textsuperscript{23}. Our previous study showed that
BECN1-mediated autophagy regulated by TXNDC17 is involved in
paclitaxel-induced drug resistance in NB cells\textsuperscript{24}.
Similar results have been observed in ovarian
cancer\textsuperscript{25}. However, the roles of TXNDC17 and BECN1 in
resistance to first-line chemotherapeutics in NB is still unknown.

Suberoylanilide hydroxamic acid (SAHA) is a broad-spectrum hydroxamic
histone deacetylase inhibitor (HDACI) that may reduce TXNDC17 expression
by upregulating the endogenous suppressor thioredoxin
(TRX)\textsuperscript{26-28}. In this study, we explored the role of
TXNDC17 in autophagy and its relationship with the resistance to
first-line chemotherapy in NB. We investigated the mechanism by which
SAHA regulates autophagy through TXNDC17 and reverses chemoresistance.

\textbf{Material and Methods}

Patients

The data of patients with NB were selected from the Therapeutically
Applicable Research to Generate Effective Treatments (TARGET) dataset
using cBioPortal (\emph{http://www.cbioportal.org.cn}). The selected
data included somatic mutations, putative copy number alterations, mRNA
expression, and patient clinical data. The\emph{BECN1} and
\emph{TXNDC17} mutation rates and types were analyzed using cBioPortal,
and the mRNA expression of \emph{BECN1} and\emph{TXNDC17} detected by
microarray was analyzed using Spearman's correlation analysis.

Cell culture

A human NB cell line SH-SY5Y was obtained from COBIOER (Nanjing,
Jiangsu, China) and cultured in MEM-F12 medium supplemented with 10\%
fetal calf serum (Gibco; Waltham, MA, USA). The cells were maintained in
a 5\% CO\textsubscript{2} incubator under saturated humidity at 37 °C,
and cells in the logarithmic growth phase were used for the experiments.

Cell proliferation and toxicity experiment

After digestion and centrifugation, an appropriate number of SH-SY5Y
cells were seeded in 96-well plates where each well contained 200 \selectlanguage{greek}μ\selectlanguage{english}l
culture medium and 1 \selectlanguage{ngerman}× 10\textsuperscript{4} cells, and each group was
repeated in triplicate wells. The plates were incubated overnight to
achieve full cell attachment. Subsequently, different doses of the drug
were added to wells at various final concentrations. After 48 h of
incubation, the medium was replaced and cell viability was measured
using the Cell Counting Kit-8 (CCK-8; APExBIO, Houston, TX, USA). After
incubation for 4 h, the absorbance was measured at 450 nm using a
multifunctional microplate detector M200 (Tecan, Zürich, Switzerland),
and cell viability was calculated according to the absorbance.

Plasmids and shRNA transfection

The TXNDC17 and the BECN1 overexpression plasmids, TXNDC17, and BECN1
shRNAs were purchased from DHbio (Guangzhou, Guangdong, China). The
overexpression plasmid and shRNA were mixed with PAX2 and pMD2.G
plasmids at a ratio of 10:7.5:3 and transfected into HEK-293T cells
using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The
lentivirus transfection solution was harvested and filtered after 24 and
48 h. SH-SY5Y cells were seeded into 6-well plates in advance, and then
1 ml lentivirus transfection solution, 1 ml culture medium, and an
appropriate amount of polybrene were added to each well. After
transfection, the medium was replaced with fresh medium. The transfected
cells were subsequently selected under puromycin with a concentration of
2 \selectlanguage{greek}μ\selectlanguage{english}g/ml for 2 weeks.

Flow cytometry

SH-SY5Y cells were seeded into 6-well plates at the appropriate density.
After exposure to the drugs for 48 h, the cells were collected and
resuspended in the binding buffer to achieve a cell density of 1 \selectlanguage{ngerman}×
10\textsuperscript{6} cells/ml. Subsequently, the cells were stained
with Annexin V-FITC/PI reagent (4Abio, Beijing, China), and single-dye
tubes were prepared. The samples were analyzed by spectral flow
cytometer SP6800 (Sony Biotechnology, San Jose, CA, USA).

Western blot

Proteins were extracted from tumor cells using RIPA buffer and the
protein concentration was estimated using the Bradford method. After
albuminous degeneration, 20 \selectlanguage{greek}μ\selectlanguage{english}g protein from each group was separated by
SDS-PAGE. The separated proteins were transferred onto PVDF membranes
and blocked with skim milk powder for 2 h. The primary antibody was
added at an appropriate dilution and the blots were incubated overnight.
Subsequently, the blots were rinsed with TBS-T three times for 15 min
each and incubated with the secondary antibody for 1 h. After rinsing
with TBS-T, the blots were stained with enhanced chemiluminescence and
visualized using a chemiluminescence imaging system (Bio-Rad
Laboratories, Hercules, CA, USA). Glyceraldehyde 3-phosphate
dehydrogenase was used as a reference in this study.

Statistical analysis

Event-free survival (EFS) was defined as the time between diagnosis and
progression, relapse, second malignancy, death from any cause, or last
follow-up contact for patients who did not experience any event. Overall
survival (OS) was defined as the time between diagnosis and death from
any cause or the last follow-up visit for live patients. OS and EFS were
estimated using the Kaplan--Meier method, and the log-rank test was used
to compare the risk of adverse events between the groups. Correlations
between factors were analyzed by a Spearman correlation analysis, and
cell viability and apoptosis rates between different groups were
assessed by Student's t-test; statistical significance was set at p
\textless{}0.05. SPSS 25.0 (IBM Corp., Armonk, NY, USA) and GraphPad
Prism 8.0 (GraphPad Software, Boston, MA, USA), were used for the
statistical analyses.

\textbf{Results}

\textbf{The mutation of \emph{BECN1} and \emph{TXNDC17} in patients with
NB was associated with poor prognosis}

In total, 1076 pediatric and adolescent patients with NB selected from
the TARGET dataset using cBioPortal were enrolled in this study. The
inclusion criteria were patients with a diagnosis of NB between 2003 and
2018, age \textless{}18 years, and clinical data uploaded to the TARGET
dataset. The median age of the patients was 8.6 years, and the
male-to-female ratio was 1.3:1. The detailed clinical features are
displayed in Table 1. The patients were enrolled in clinical trials,
including ANBL00B1, ANBL0532, ANBL0032, and AEPI07N1. The commonly used
induction chemotherapy regimens included CTX, doxorubicin, and
vincristine, alternated with DDP and VP16. The 10-year EFS rate of the
whole group was 36.9\% \selectlanguage{ngerman}± 1.5\%, and the 10-year OS rate was 57.5\% ±
1.8\%. The mutation frequencies of \emph{BECN1} and \emph{TXNDC17} were
6.80\% and 2.66\%, respectively (Figure 1A). The mRNA expression
of\emph{BECN1} was associated with \emph{TXNDC17} (\emph{P} = 1.48 ×
10\textsuperscript{-14}), and the Spearman correlation coefficient was
0.46 (Figure 1B). The 10-year EFS rates for the patients with no
mutations of \emph{BECN1} and \emph{TXNDC17} (n = 984), mutation
of\emph{BECN1} (n = 70), and mutation of \emph{TXNDC17} (n = 29) were
37.1\% ± 1.5\%, 34.5\% ± 8.8\%, and 24.3\% ± 5.1\%, respectively
(\emph{P} \textless{}0.05, Figure 1C); the 10-year OS rates were 58.0\%
± 1.8\%, 37.4\% ± 9.1\%, and 41.4\% ± 5.9\%, respectively
(\emph{P}\textless{}0.05, Figure 1D).

Table 1. Characteristics of the patients with neuroblastoma.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Characteristics & Cases (n) & Percent (\%)\tabularnewline
\midrule
\endhead
Sex & &\tabularnewline
Male & 613 & 57\tabularnewline
Female & 463 & 43\tabularnewline
Race & &\tabularnewline
White & 792 & 74\tabularnewline
Others & 284 & 26\tabularnewline
Ethnicity & &\tabularnewline
Not Hispanic or Latino & 870 & 81\tabularnewline
Hispanic or Latino & 206 & 19\tabularnewline
INSS stage & &\tabularnewline
Stage I & 89 & 8\tabularnewline
Stage II & 61 & 6\tabularnewline
Stage III & 94 & 9\tabularnewline
Stage IV & 777 & 72\tabularnewline
Stage IVS & 55 & 5\tabularnewline
Primary site & &\tabularnewline
Abdomen & 827 & 77\tabularnewline
Pelvic & 15 & 1\tabularnewline
Chest & 106 & 10\tabularnewline
Head and neck & 21 & 2\tabularnewline
Others & 107 & 10\tabularnewline
\bottomrule
\end{longtable}

INSS, International Neuroblastoma Staging System.

\textbf{Upregulation of BECN1 or TXNDC17 resulted in decreased
sensitivity to DDP, VP16, and CTX in NB}

In our previous study, we demonstrated that resistance to DDP, VP16, and
CTX in NB was induced by autophagy, and this process was associated with
BECN1\textsuperscript{15}. BECN1\textsuperscript{high},
BECN1\textsuperscript{low}, TXNDC17\textsuperscript{high}, and
TXNDC17\textsuperscript{low} SH-SY5Y cells were obtained by up- and
downregulating BECN1 and TXNDC17. Negative control (NC),
BECN1\textsuperscript{high}, BECN1\textsuperscript{low},
TXNDC17\textsuperscript{high}, and TXNDC17\textsuperscript{low} cells
were exposed to DDP, VP16, and CTX at the same concentration for the
same time. The respective cell proliferation rates of
BECN1\textsuperscript{high}, NC, and BECN1\textsuperscript{low} were
74\% vs. 62\% vs. 59\% after exposure to 2 \selectlanguage{greek}μ\selectlanguage{english}M DDP, 76\% vs. 54\% vs.
55\% with 250 nM VP16, and 81\% vs. 73\% vs. 72\% with 2 mM CTX (Figure
2A, 2B, 2C). These results show that BECN1 overexpression results in
lower sensitivity to DDP, VP16, and CTX in NB cells. While the
respective cell proliferation rates of TXNDC17\textsuperscript{high},
NC, and TXNDC17\textsuperscript{low} were 82\% vs. 61\% vs. 50\% with 2
\selectlanguage{greek}μ\selectlanguage{english}M DDP, 77\% vs. 51\% vs. 20\% with 250 nM VP16, and 84\% vs. 73\% vs.
33\% with 2 mM CTX (Figure 2D, 2E, 2F). These results indicated that the
upregulation of TXNDC17 also reduced the sensitivity of NB cells to DDP,
VP16, and CTX.

\textbf{BECN1-mediated autophagy was regulated by TXNDC17 in NB}

The levels of autophagy-related proteins in
BECN1\textsuperscript{high}and BECN1\textsuperscript{low} cells were
detected by western blot, and the results showed that the levels of
LC3B-II increased and those of P62 decreased, indicating that autophagy
was enhanced in BECN1\textsuperscript{high} cells. However, the levels
of LC3B-II decreased, and P62 increased, indicating that autophagy was
decreased in BECN1\textsuperscript{low} cells (Figure 3A). These results
suggested that autophagy in the NB was mediated by BECN1. Western blot
was performed to detect autophagy-related proteins in the
TXNDC17\textsuperscript{high} and TXNDC17\textsuperscript{low} cells.
The results showed high levels of LC3B-II and low levels of P62 in the
TXNDC17\textsuperscript{high} cells, indicating enhanced autophagy. The
levels of LC3B-II decreased, and P62 increased in
TXNDC17\textsuperscript{low} cells, indicating that autophagy was
inhibited (Figure 3A). These results show that TXNDC17 is involved in
the regulation of autophagy in NB cells and interacts with BECN1.

To further clarify the relationship between TXNDC17 and BECN1,
BECN1\textsuperscript{high}TXNDC17\textsuperscript{low} and
BECN1\textsuperscript{low}TXNDC17\textsuperscript{high} cells were
obtained by downregulating TXNDC17 in BECN1\textsuperscript{high} cells
and upregulating TXNDC17 in BECN1\textsuperscript{low} cells. Compared
to BECN1\textsuperscript{high} cells, the levels of BECN1, LC3B-II, and
TXNDC17 were lower, and P62 was higher in
BECN1\textsuperscript{high}TXNDC17\textsuperscript{low} cells. Compared
to BECN1\textsuperscript{low} cells, the levels of BECN1, LC3B-II, and
TXNDC17 were higher, and P62 was lower in
BECN1\textsuperscript{low}TXNDC17\textsuperscript{high} cells (Figure
3B). These results indicate that BECN1-mediated autophagy is regulated
by TXNDC17 in NB.

\textbf{SAHA inhibited autophagy by decreasing the expression of
TXNDC17}

The IC50 of SH-SY5Y cells to SAHA was 2 \selectlanguage{greek}μ\selectlanguage{english}M, which was detected by the
Cell Counting Kit-8 method. TXNDC17\textsuperscript{high} and NC cells
were exposed to 1 \selectlanguage{greek}μ\selectlanguage{english}M SAHA for 48 h, and western blot was used to detect
autophagy-related proteins. Compared to the NC group, the levels of
LC3B-II, TXNDC17, and BECN1 decreased, and P62 increased in
TXNDC17\textsuperscript{high} cells after exposure to SAHA (Figure 4A).
SAHA effectively inhibited the expression of TXNDC17 and then inhibited
autophagy. SH-SY5Y cells were exposed to DDP, VP16, or CTX, with or
without SAHA, for 48 h. The levels of LC3B-II, TXNDC17, and BECN1
decreased in the group treated with SAHA (Figure 4B). This suggests that
DDP, VP16, and CTX combined with SAHA can effectively inhibit
autophagy-induced chemoresistance.

\textbf{SAHA enhanced the sensitivity of NB to DDP, VP16, and CTX}

To determine the effects of SAHA combined with chemotherapeutics,
SH-SY5Y cells were exposed to different concentrations of DDP, VP16, and
CTX with or without the addition of 0.5 \selectlanguage{greek}μ\selectlanguage{english}M SAHA. The proliferation rate
of cells exposed to SAHA was 90\%, while those of cells treated with
DDP, VP16, or CTX alone were 53\%, 51\%, and 74\%, respectively. The
cell proliferation rates after combination treatment with SAHA were
32\%, 27\%, and 27\%, respectively. This indicated that SAHA could only
slightly inhibit the growth of tumor cells when used alone, and the
inhibitory effect of chemotherapeutics was significantly enhanced when
combined with SAHA (Figure 5A, 5B, 5C). SAHA enhanced the sensitivity of
NB cells to DDP, VP16, and CTX.

\textbf{SAHA enhanced chemotherapy-induced apoptosis in NB}

To further explore the additional mechanisms of SAHA combined with
chemotherapeutics in killing NB cells, SH-SY5Y cells were exposed to 5
\selectlanguage{greek}μ\selectlanguage{english}M DDP, 500 nM VP16, and 5 mM CTX with or without the addition of 0.5 \selectlanguage{greek}μ\selectlanguage{english}M
SAHA for 48 h. Apoptosis was analyzed using flow cytometry. The
apoptotic rates of SH-SY5Y cells exposed to DDP, VP16, or CTX alone were
23.1\%, 26.4\%, and 11.0\%, respectively. The apoptosis rates increased
to 41.4\%, 50.0\%, and 20.6\%, respectively, after combination treatment
with SAHA. Compared to treatment with chemotherapeutics or SAHA alone,
cell apoptosis was significantly enhanced after combination treatment
with SAHA (Figure 6A, 6B, 6C). These results indicated that SAHA also
enhanced the apoptosis of NB, in addition to improving the sensitivity
to chemotherapeutics.

\textbf{Discussion}

NB is sensitive to initial chemotherapy, but is prone to acquired drug
resistance. This study investigated the mechanism of drug resistance to
first-line chemotherapeutics in NB, which indicated that TXNDC17 and
BECN1 were associated with poor prognosis and that TXNDC17-mediated
autophagy played an important role in resistance to chemotherapeutics.
Autophagy is a pathway of cellular tolerance to stress that is
associated with several physiological and pathological states, such as
intracellular circulation, nutrient starvation, and, most importantly,
chemotherapy\textsuperscript{29, 30}. In several tumor types, autophagy
protects tumor cells against stress, leading to
chemoresistance\textsuperscript{31-33}. Our previous studies on NB cells
have shown that autophagy induces primary resistance to
paclitaxel\textsuperscript{24}, and acquired resistance to DDP, VP16,
and CTX, which are commonly used induction chemotherapeutics in North
American and European regimens for patients with high-risk
NB\textsuperscript{1}.

To further clarify the relationship between autophagy and
chemoresistance in NB, BECN1 expression was down- and upregulated in
this research. The results showed that the overexpression of BECN1
enhanced autophagy and significantly reduced the sensitivity of NB cells
to DDP, VP16, and CTX. Enhanced BECN1-mediated autophagy is one of the
reasons for the resistance to DDP, VP16, and CTX. Previous studies have
shown that BECN1-mediated autophagy is associated with leukemia
resistance to CTX\textsuperscript{34}; lung cancer, embryonal carcinoma,
cervical carcinoma resistance to DDP\textsuperscript{35-37}; and
cervical cancer resistance to VP16\textsuperscript{38}. These findings
suggest that BECN1-mediated autophagy is a common factor in chemotherapy
resistance, thus adversely affecting the prognosis of multiple tumor
types, including NB.

This finding was confirmed in patients with NB. We performed a survival
analysis of 1076 patients with NB enrolled in the TARGET public database
and found that the 10-year EFS and OS rates of patients with\emph{BECN1}
mutations were poorer than those of patients without\emph{BECN1}
mutations. BECN1 was involved in the regulation of the occurrence and
development of NB, and had a similar effect in protecting and promoting
the growth of tumor cells.

However, BECN1 is regulated by several cellular signals that ultimately
regulated autophagy \textsuperscript{22, 39, 40}. In the survival
analysis of the enrolled patients, the 10-year EFS and OS rates were
significantly lower in the \emph{TXNDC17} mutation group. Both TXNDC17
and BECN1 are significantly involved in the chemoresistance of NB. A
Spearman correlation analysis revealed that the mRNA expression levels
of \emph{TXNDC17} and \emph{BECN1} were positively correlated. Our
previous study showed that TXNDC17 is associated with primary resistance
to paclitaxel in NB\textsuperscript{24}. Moreover, TXNDC17 promotes
tumor resistance to paclitaxel in ovarian cancer by regulating
BECN1-mediated autophagy, and high TXNDC17 expression has been confirmed
to be associated with poor prognosis in patients with ovarian
cancer\textsuperscript{25}.

To further explore the relationship between TXNDC17 and BECN1 in
autophagy-related chemoresistance, TXNDC17 was down- and upregulated
using genetic engineering. BECN1 levels decreased and autophagy was
inhibited after TXNDC17 downregulation. In validation experiments,
autophagy in downregulated-BECN1 NB cells was enhanced by upregulating
TXNDC17. Thus, TXNDC17 regulates autophagy by regulating BECN1
expression in NB.

Previous studies have shown that the transcription factor NF-\selectlanguage{greek}κ\selectlanguage{english}B drives
the expression of BECN1\textsuperscript{41}, while TXNDC17 inhibits
tumor necrosis factor-induced NF-\selectlanguage{greek}κ\selectlanguage{english}B activation. DYNLL1, which was the
only identified potential substrate of TXNDC17, could interact with the
inhibitors of NF-\selectlanguage{greek}κ\selectlanguage{english}B\textsuperscript{42}. In addition, the NF-\selectlanguage{greek}κ\selectlanguage{english}B1 family
member RELA upregulated the mRNA and protein levels of BECN1 in
different cellular systems through the activation of a conserved NF-\selectlanguage{greek}κ\selectlanguage{english}B1
binding site in the promoter of the
\emph{BECN1}gene\textsuperscript{43}. Therefore, we hypothesized that
TXNDC17 regulates BECN1-mediated autophagy through the NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway.
This study also found that the overexpression of TXNDC17 can reduce the
sensitivity of NB cells to DDP, VP16, and CTX, leading to drug
resistance, which has an effect similar to that of BECN1.

As a broad-spectrum HDACI, SAHA upregulates the expression of TRX
inhibitors in various tumor cells, thereby inhibiting TRX
activity\textsuperscript{28}. TXNDC17 is a member of the TRX family.
This study showed that TXNDC17, along with its related autophagy, was
specifically inhibited by SAHA in NB. After the combination of
chemotherapeutics and SAHA, autophagy decreased, and NB was
significantly more sensitive to DDP, VP16, and CTX. These results
suggest that autophagy-induced chemoresistance can be reversed by
combining SAHA with chemotherapy.

Moreover, this study found that SAHA can promote the apoptosis of NB
cells, which again proves that SAHA has multiple antitumor effects on
NB. However, the cell inhibition rate was much greater than the
apoptosis rate in NB cells exposed to the same concentration and
treatment time as the chemotherapeutics, indicating that apoptosis
accounted for only a small proportion of the killing activity of SAHA.
Therefore, we considered this to have little impact on the conclusions
drawn from this study. Whether there is a synergy between the molecular
mechanisms of autophagy and apoptosis is worth further investigation.

In conclusion, this study confirmed that enhanced BECN1-mediated
autophagy was related to the resistance of NB cells to DDP, VP16, and
CTX in a first-line induction chemotherapy regimen. SAHA reduces BECN1
expression by inhibiting TXNDC17, thereby reversing chemotherapy
resistance and improving the sensitivity of NB cells to chemotherapy.
These findings should be investigated further to improve the efficacy of
NB treatment.

\textbf{Disclosure:}

\textbf{Funding Information}

This work was supported by a grant from the GuangDong Basic and Applied
Basic Research Foundation (2023A1515010145). The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.

\textbf{Conflict of Interest}

The authors have no conflict of interest.

\par\null

\textbf{Ethics Statement}

Approval of the research protocol by an Institutional Reviewer Board:
N/A

Informed Consent: N/A

Registry and the Registration No. of the study/trial: N/A

Animal Studies: N/A

\textbf{Author Contributions}

ZZ designed this study. CZ, ZL, and ZW conducted the main experiments.
JW, JH, FS, JZ, and SL supervised the research. TC contributed to
technical Support. TC and ZL helped to analyze data. CZ wrote the
manuscript. All authors read and approved the final manuscript

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\textbf{Figure legends:}

Figure 1. The prognosis for patients with mutation of BECN1 or TXNDC17.

Mutation frequency and specific mutation types of \emph{BECN1}
and\emph{TXNDC17} in the sample data. (B) Correlation analysis between
mRNA expression of \emph{BECN1} and \emph{TXNDC17} . (C) Patients with
mutation of \emph{BECN1} (\emph{P} = 0.001) or \emph{TXNDC17}(\emph{P} =
0.04) have lower 10-year EFS rates compared to the unaltered group. (D)
Patients with mutation of \emph{BECN1} (\emph{P}= 0.005) or
\emph{TXNDC17} (\emph{P} = 0.0001) have lower 10-year OS rates compared
to the unaltered group.

Figure 2. Upregulation of BECN1 or TXNDC17 results in decreased
chemosensitivity in NB cells.

Upregulation of BECN1 results in decreased chemosensitivity and
downregulation of BECN1 led to increased chemosensitivity in NB cells
exposed to DDP (A), VP16 (B), and CTX (C). Upregulation of TXNDC17
results in decreased chemosensitivity and downregulation of TXNDC17
leads to increased sensitivity in NB cells exposed to DDP (D), VP16 (E),
and CTX (F). * \emph{P} \textless{}0.05, * * \emph{P} \textless{}0.01, *
* * \emph{P} \textless{}0.001, two-sided t-test.

Figure 3. BECN1-mediated autophagy is regulated by TXNDC17 in NB

(A) The level of LC3B-II decreases, and that of P62 increases after the
downregulation of BECN1. The level of LC3B-II increases, and that of P62
decreases after the upregulation of BECN1. The level of LC3B-II
decreases, and that of P62 increases after the downregulation of
TXNDC17. The level of LC3B-II increases, and P62 decreases after the
upregulation of TXNDC17. (B) Compared with
BECN1\textsuperscript{high}cells, the levels of BECN1, LC3B-II, and
TXNDC17 decrease, and that of P62 increases in
BECN1\textsuperscript{high}TXNDC17\textsuperscript{low}cells. Compared
with BECN1\textsuperscript{low} cells, the levels of BECN1, LC3B-II, and
TXNDC17 increase, and that of P62 decreases in
BECN1\textsuperscript{low}TXNDC17\textsuperscript{high} cells.

Figure 4. SAHA inhibits autophagy by decreasing the expression of
TXNDC17

(A) SH-SY5Y cells with upregulated TXNDC17 show decreased LC3B-II,
TXNDC17, and BECN1 levels and increased P62 after exposure to SAHA. (B)
Compared with using DDP, VP16, and CTX alone, the levels of LC3B-II,
TXNDC17, and BECN1 decrease, and that of P62 increases after combining
SAHA.

Figure 5. SAHA can enhance the sensitivity of NB to chemotherapy

(A) Compared with using DDP alone, the cell proliferation rate is
significantly reduced after combining SAHA. (B) Compared with using VP16
alone, the cell proliferation rate is significantly reduced after
combining SAHA. (C) Compared with using CTX alone, the cell
proliferation rate is significantly reduced after combining SAHA.
*\emph{P} \textless{}0.05, * * \emph{P} \textless{}0.01, * * *
\emph{P}\textless{}0.001, two-sided t-test.

Figure 6. SAHA can enhance chemotherapy-induced apoptosis in NB

(A) Compared with exposure to DDP alone, the apoptosis rate is enhanced
in the DDP + SAHA group. (B) Compared with exposure to VP16 alone, the
apoptosis rate is enhanced in the VP16 + SAHA group. (C) Compared with
exposure to CTX alone, the apoptosis rate is enhanced in the CTX + SAHA
group. * \emph{P} \textless{}0.05, * * \emph{P} \textless{}0.01, * *
*\emph{P} \textless{}0.001, two-sided t-test.\selectlanguage{english}
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