GOLGA8B is a critical gene for prostate cancer progression and resistance of

castration-resistant prostate cancer to cabazitaxel

1.Introduction
1. Introduction

Prostate cancer (PCa) commonly occurs in older men with an incidence rate of more
than 20% and has become the second leading cause of cancer-related death among
men in the United States of America(1). The morbidity and mortality rates associated
with PCa are also rapidly increasing in China(2). Currently, there are many effective
methods for treating PCa, with androgen deprivation therapy (ADT) being the
preferred first-line treatment(3). However, after a period of ADT, patients with
PCainevitably develop castration-resistant prostate cancer (CRPC)(4). CRPC can be
treated with many therapeutic methods, including chemotherapy(5). Taxane class of
chemotherapeutic drugs, including docetaxel, has been used for treating CRPC(6).
However, patients with CRPC treated with docetaxel eventually become resistant to
the drug, requiring intervention with other types of taxane drugs to treat docetaxel-
resistant CRPC.
Prostate cancer (PCa) is a widespread malignancy, primarily affecting older men, with
a prevalence rate exceeding 20%. It is the second most frequent cause of cancer-
related death in men within the United States America (1).China is also witnessing a
rapid increase in PCa incidence and mortality rates (2).Currently, a range of effective
treatments present for PCa, with androgen deprivation therapy (ADT) being the
favored initial approach(3).However, after undergoing androgen deprivation therapy
(ADT), patients consistently advance to develop castration-resistant prostate cancer
(CRPC)(4).CRPC can be managed through various therapeutic modalities, including
chemotherapy (5). Taxane-based chemotherapeutic agents such as docetaxel has been
used in CRPC treatment(6). However, patients with CRPC treated with docetaxel
eventually develop resistance to the drug, necessitating the use of alternative taxane
drugs to address docetaxel-resistant CRPC.
Cabazitaxel is a member of the taxane family of drugs. It can bind tubulin and
promote microtubule assembly by stabilizing them and preventing their
depolymerization, which consequently interferes with cell division and results in cell
death(7, 8). Cabazitaxel has been shown to exert cytotoxic activity and retard the
growth of many docetaxel-resistant tumor cells(9). Furthermore, the roles of
cabazitaxel in inhibiting human xenograft tumor growth(10) and in treating CRPC(11,
12) have been reported. Despite the efficacy of cabazitaxel in treating CRPC, patients
ultimately become resistant to cabazitaxel, but the mechanism underlying this
resistance remains unclear.
Cabazitaxel belongs to the taxane family of drugs. It can bind tubulin and promote
microtubule assembly by stabilizing them and preventing their depolymerization,
which consequently interferes with cell division and results in cell death(7, 8).
Cabazitaxel has been shown to exert cytotoxic activity and retard the growth of many
docetaxel-resistant tumor cells(9). Furthermore, the roles of cabazitaxel in inhibiting
human xenograft tumor growth(10) and in treating CRPC(11, 12) have been reported.
Despite the efficacy of cabazitaxel in treating CRPC, patients ultimately become
resistant to cabazitaxel, but the mechanism underlying this resistance remains unclear.
Genetic alterations play an important role in resistance to chemotherapeutics drugs in
PCa. Many studies have shown that genetic alterations may be a critical reason for
resistance to chemotherapy drugs like docetaxel and vinblastine in PCa(13-15).
Additionally, studies have found that some gene mutations can cause PCa resistance
to cabazitaxel(16, 17). Hence, investigating altered gene expression can help elucidate
the mechanism of CRPC resistance to cabazitaxel. In this study, using the GSE158494
dataset from the Gene Expression Omnibus (GEO) database, we identified the
upregulated genes in cabazitaxel-resistant CRPC cells. Our study revealed that these
genes can influence the progression of PCa, and the GOLGA8Bgene can even affect
the prognosis of patients with PCa. GOLGA8B was also found to be upregulated in
clinical PCa and CRPC samples and affect the sensitivity of CRPC cells to cabazitaxel
and docetaxel. Our study provides evidence for the important role of GOLGA8B in
PCa development and CRPC resistance to cabazitaxel.
Genetic alterations have emerged as significant contributors to resistance against
chemotherapeutic drugs in prostate cancer (PCa).Many studies have shown the pivotal
role of genetic alterations as a fundamental factor behind chemotherapy resistance in
PCa, notably for drugs like docetaxel and vinblastine (13-15). Furthermore, research
has highlighted that certain gene mutations can cause resistance to cabazitaxel in PCa
(16, 17). Consequently, investigating changes in gene expression becomes crucial for
unraveling the mechanisms underpinning castration-resistant prostate cancer (CRPC)
resistance to cabazitaxel. In the present study, the GSE158494 dataset obtained from
the Gene Expression Omnibus (GEO) database shows the overexpressed genes in
cabazitaxel-resistant CRPC cells. The impact of these genes on prostate cancer
progression, with particular emphasis on the role of the GOLGA8B gene
in influencing the prognosis of PCa patients. Notably, GOLGA8B was identified as
being upregulated in clinical PCa and CRPC specimens, demonstrating its influence
on the sensitivity of CRPC cells to both cabazitaxel and docetaxel. This investigation
reinforces the pivotal role of GOLGA8B in the development of PCa and its resistance
to cabazitaxel
2.Materials and methods
2.Methodology

2.1 Data collection

2.1Collection of Data.
This study encompassed the analysis of multiple datasets retrieved from various
sources. Firstly, the GSE158494 dataset utilized , which includes RNA-sequence data
derived from cabazitaxel-sensitive and -resistant DU145 and PC-3 CRPC cells.
Additionally, two more datasets, GSE33455 and GSE36135, were acquired, both of
which comprise sequence data from docetaxel-resistant CRPC cells. These datasets
were sourced from the Gene Expression Omnibus (GEO) database
(http://www.ncbi.nlm.nih.gov/geo/).Furthermore, incorporated analysis of GSE35988
dataset, providing valuable information pertaining to patients diagnosed with PCa and
CRPC. In addition to these GEO datasets, it is also examined patient data from two
distinct databases: The Cancer Genome Atlas (TCGA) can be found at
http://cancergenome.nih.gov/, while the Chinese Prostate Cancer Genome and
Epigenome Atlas (CPGEA) is accessible through http://www.cpgea.com
2.2 Data handling

2.2 Processing of data

The raw sequence data from the different databases were normalized using the R
software “limma” package; the probe ID was substituted to the corresponding gene
ID. The genes with |log2FC > 1| and P< 0.05 were considered cabazitaxel resistance-
related genes. We also obtained clinical data from different databases.
Raw sequence data retrieved from various databases underwent normalization using
the "limma" package in the R software. During this process, the probe ID was
matched to the respective gene ID. Genes meeting the criteria of |log2FC > 1| and P<
0.05 were classified as cabazitaxel resistance-related genes. Additionally, clinical data
were obtained from diverse databases

2.3 Pathway analysis

Pathway analysis was conducted through gene ontology (GO) and Kyoto
Encyclopedia of Genes and Genomes (KEGG) analysis using the online tool
Metascape (http://metascape.org/), and the bubble map was constructed using the R
software “ggplot2” package.

Pathway analysis was conducted through the utilization of GO and KEGG. The online
tool Metascape, accessible at http://metascape.org/, was employed for this analysis.
Following the analysis, bubble plots were generated using the "ggplot2" package
within the R software.
2.4 Survival analysis

The online web tool “GEPIA” was used to examine the correlation between
cabazitaxel resistance-related genes and the disease-free survival (DFS) status in
patients with PCa. Univariate and multivariate Cox regression analyses were
performed to identify the proper terms to build the nomogram. A forest plot was used
to show the P value, HR, and 95% CI of each variable through the R software
‘forestplot’ package. A nomogram was developed based on the results of multivariate
Cox proportional hazards analysis to predict the overall recurrence.
The online web tool "GEPIA" was utilized to facilitate the exploration of correlations
between genes associated with cabazitaxel resistance and the disease-free survival
(DFS) status in prostate cancer (PCa) patients. Univariate and multivariate Cox
regression analysis was performed to determine the variables necessary to create the
nomogram.Additionally, the 'forest plot' package in the R software was employed to
display P values, hazard ratios (HR), and 95% (CI) for each variable using a forest
plot.
Subsequently, a nomogram was constructed based on the outcomes of the multivariate
Cox proportional hazards analysis,serving as a predictive tool for overall recurrence

2.5 Immune infiltration analysis

The relationship between the expression of cabazitaxel resistance-related genes and

immune cell infiltration were analyzed using Spearman correlation based on the data
from the TCGA database.
Using data from the TCGA database, Spearman correlation analysis was used to
evaluate the relationship between the expression of cabazitaxel resistance-related
genes and the immune response.

2.6 Clinical specimen collection

2.6 Collection of Clinical Specimens

Clinical PCa and CRPC specimens were collected from Tongji Hospital, School of
Medicine, Tongji University. The collection method was approved by the Ethics
Committee of Tongji Hospital, School of Medicine, Tongji University (SBKT-2021-
220). Patients who provided the samples were informed of the experiment and gave
informed consent.
Specimens were obtained from individuals who had been diagnosed with PCa and
CRPC at Tongji Hospital, School of Medicine, Tongji University. The collection
procedure obtained official approval from the Ethics Committee of Tongji Hospital,
School of Medicine, Tongji University, under Approval No (SBKT-2021-220).
Comprehensive information about the study was provided to all participating patients,
and they willingly gave informed consent for the utilization of their samples in the
research.

2.7 Cell culture and drug exposure treatment

PCa cell lines were purchased from the Chinese Academy of Science Cell Bank
(Shanghai, China). The human CRPC cell lines PC-3 and DU145 were cultured in
Roswell Park Memorial Institute (RPMI) 1640 medium (Catalog No. R8758, Sigma,
Darmstadt, Germany) containing 10% fetal bovine serum (FBS) (Catalog No. 10091,
Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in a humid environment with
5% CO2 and 95% air at 37°C. Cabazitaxel (Catalog No. S3022) and docetaxel
(Catalog No. S1148) were purchased from SelleckChem (Houston, TX, USA). The
CRPC cells were treated with cabazitaxel or docetaxel (2 nmol/L) for 24 h.
PCa cell lines were obtained from the Chinese Academy of Science Cell Bank
(Shanghai, China). The human CRPC cell lines PC-3 and DU145 were specifically
grown in Roswell Park Memorial Institute (RPMI) 1640 media (Sigma, Darmstadt,
Germany, Catalog No. R8758) supplemented with 10% foetal bovine serum (FBS)
(Gibco, Thermo Fisher Scientific, Waltham, MA, USA, Catalog No. 10091). Cell
cultures were kept viable at a temperature of 37°C in a humidified atmosphere with
5% CO2 and 95% air. Docetaxel and cabazitaxel were purchased for the study
(SelleckChem, Houston, TX, USA, Catalog No. S1148 and S3022),respectively. For
24 hours, the CRPC cells were treated to docetaxel or cabazitaxel at a dose of 2
nmol/L.

2.8 Cell transfection and lentivirus production

2.8 Lentivirus Production and Cellular Transfection

Cell transfection assays were performed using Lipofectamine 2000 (Catalog No.
11668019, Thermo Fisher Scientific). shGOLGA8B lentivirus was constructed for
specific gene knockdowns. The gene-specific shRNAs and control lentivirus
(shControl) were purchased from Youze Biotechnology Company (Hunan, China).
Cell transfection experiments were conducted employing Lipofectamine 2000
(Thermo Fisher Scientific, Catalog No. 11668019). Specifically, shGOLGA8B
lentivirus was designed to facilitate targeted gene knockdown. The gene-specific
shRNAs and a control lentivirus (shControl) were procured from Youze
Biotechnology Company, located in(Hunan, China).

2.9 RNA extraction and qRT-PCR procedure

The total RNA was extracted from CRPC cell lines using TRIzol Reagent (Sigma–
Aldrich, St. Louis, MO, USA, Catalog No. T9424). cDNA was transcribed using a
reverse transcription kit (Advantage® RT-for-PCR Kit, Takara Bio Inc., Kusatsu,
Japan, Catalog No. 639505). Finally, gene expression was analyzed using a real-time
PCR kit (TB Green® Premix Ex Taq™ II, Takara Bio Inc., Catalog No. RR420A)
according to the manufacturer’s instructions. The PCR primers used for GOLGA8B
and GAPDH (reference gene) are shown in Table 1. The 2−ΔΔCt method was used to
quantify mRNA expression levels.
Using the TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA, catalog number
T9424), total RNA was isolated from CRPC cell lines. Then, using a reverse transcript
kit (Advantage® RT-for-PCR Kit, Takara Bio Inc., Kusatsu, Japan, Catalog No.
639505), cDNA synthesis was carried out. finally, via a real-time PCR kit (TB
Green® Premix Ex Taq™ II, Takara Bio Inc., Cat. No. RR420A), gene expression
analysis was carried out according to the directions provided by the manufacturer.
Table 1 provides the PCR primer sequences for the reference genes GOLGA8B and
GAPDH. By using the 2−ΔΔCt technique, mRNA expression was quantified
.
2.10 Antibodies

Rabbit polyclonal anti-GOLGA8B antibody (Catalog No. ab155806) and Rabbit

monoclonal anti-GAPDH antibody (Catalog No. ab9485) were purchased from
Abcam (Abcam UK, Cambridge, UK).
Abcam (Abcam UK, Cambridge, UK) supplied the rabbit polyclonal anti-GOLGA8B
antibody (Catalogue No. ab155806) and the rabbit monoclonal anti-GAPDH
antibody (Catalogue No. ab9485).

2.11 Western blotting

Tissue samples and cell line proteins were extracted using RIPA lysis buffer. Protein
samples were treated with Dual Colour Protein Loading Buffer (Thermo Fisher
Scientific, Waltham, MA, USA). Sodium dodecyl-sulfate polyacrylamide gel
electrophoresis (10%) was used to separate proteins, which were then transferred onto
nitrocellulose membranes (Merck KGaA, Darmstadt, Germany). Protein-Free Rapid
Blocking Buffer (Thermo Fisher Scientific) was utilized to block the membranes.
Then, the membranes were incubated at 4°C overnight with primary antibodies
against GOLGA8B (1:1000) and GAPDH (1:1000) (Abcam UK, Cambridge, UK).
The next day, the membranes were washed thrice using 1× TBST (10 min/cycle) and
then incubated at room temperature for 1.5 h with a matched secondary antibody
(Catalog No. A0208, HRP-labeled Goat Anti-Human IgG (H+L), Beyotime
Biotechnology, Shanghai, China). Finally, the membranes were exposed to X-ray
film.

Proteins were extracted from tissues and cell lines using RIPA lysis buffer. Following
extraction, protein samples were processed with( Thermo Fisher Scientific Waltham,
MA, USA) Dual Colour Protein Loading Buffer. Protein separation was achieved
through SDS-PAGE using a 10% gel, followed by protein transfer onto nitrocellulose
membranes sourced from (Merck KGaA in Darmstadt, Germany).The (Thermo Fisher
Scientific) Protein-Free Rapid Blocking Buffer used to block the membranes. The
membranes were next treated with primary antibodies against GOLGA8B (dilution
1:1000) and GAPDH (dilution 1:1000) acquired from (Abcam UK,Cambridge, UK)
incubated overnight at 4°C. The membranes were then incubated at room temperature
for 1.5 hours with the corresponding secondary antibody (Catalogue No. A0208,
HRP-labeled Goat Anti-Human IgG (H+L), obtained from Beyotime Biotechnology in
Shanghai, China) the following day after being washed three times with 1xTBST (10
min/cycle). Finally, by exposing the membranes to X-ray film, protein identification
was accomplished

2.12 Cell proliferation assay

2.12 Assessment of cell proliferation

Cell proliferation ability was determined using Cell counting kit-8 (CCK-8) (Dojindo,
Japan). Briefly, cells were placed in 96-well plates (3000 cells/well) and cultured with
200 µL RPMI 1640+10% FBS for 0, 24, 48, or 72 h. After culturing, cells were
detected using CCK-8, following the manufacturer’s instructions. Absorbance at 450
nm was measured using a spectrophotometer (LD942, Beijing, China).
The Cell Counting Kit 8 (CCK 8) from (Dojindo, Japan), was used to assess the
potential for cell growth prolifieration.Briefly, 3000 cells/well of 96-well plates were
used for the first inoculation of cells. Then, they were cultivated for a variety of
lengths of time (0, 24, 48, or 72 h) in 200uL of RPMI 1640 supplemented with 10%
FBS. Cells were submitted to the CCK8 test following the incubation time in
accordance with the manufacturer's instructions. A spectrophotometer (LD942,
Beijing, China) was used to measure the absorbance at 450 nm

2.13 Statistical analysis

The matrix data were analyzed using R version 4.0.3 (Institute for Statistics and
Mathematics, Vienna, Austria; https://www.r-project.org). Comparisons between two
groups were performed using the Wilcoxon test and among more than two groups
using the Kruskal–Wallis test. Hazard ratios (HRs), 95% confidence interval (95%
CI), and P values were used as statistical metrics. Two-tailed, P< 0.05 was considered
statistically significant.
R version 4.0.3 (Institute for Statistics and Mathematics, Vienna, Austria;
https://www.r-project.org) was used to analyze the matrix data. The Wilcoxon test
used to compare two groups, and the Kruskal-Wallis test for comparisons involving
more than two groups. P values, 95% confidence intervals, and hazard ratios (HRs)
were among the statistical measures evaluated. Considering both tails, a significance
level of P< 0.05 was regarded as indicating statistical significance.

3.Results

3.1 Ten hub genes upregulated in cabazitaxel-resistant CRPC cells

3.1 Identification of Ten Upregulated Hub Genes in Cabazitaxel-Resistant CRPC
Cells

We first attempted to identify the critical genes involved in the occurrence of CRPC
resistance to cabazitaxel using the GSE158494 dataset, which included the sequence
data of cabazitaxel-sensitive and -resistant DU145 and PC-3 CRPC cells. We
constructed volcano maps to reflect the differentially-expressed genes between
cabazitaxel-sensitive and -resistant CRPC cells (Figure 1A, B). As a result, ten hub
genes were found to be upregulated in cabazitaxel-resistant cells in comparison to the
sensitive cells; interestingly, no downregulated genes were identified (Figure 1C).

To uncover the critical genes involved in the development of CRPC resistance to

cabazitaxel, analysis was conducted using the GSE158494 dataset. This dataset
comprises sequencing data from both cabazitaxel-sensitive and -resistant DU145 and
PC-3 CRPC cells. The generation of volcano plots facilitated the visualization of
differential gene expression between cabazitaxel-sensitive and -resistant CRPC cells
(Figure 1A, B). Remarkably, this analysis revealed the upregulation of ten hub genes
in cabazitaxel-resistant cells, with no detection of downregulated genes (Figure 1C).

3.2 Enriched pathways associated with cabazitaxel resistance-related hub genes

Next, we aimed to identify the pathways associated with these cabazitaxel res
istance-related genes using both GO analysis and KEGG pathway analysis. We built
bubble maps to reflect the pathways in which these genes are enriched. In the GO
analysis, we found that these genes were mainly enriched in the humoral immune
response pathway (Figure 2A). In the KEGG pathway analysis, we found that these
genes were mainly enriched in the IL-17 signaling pathway (Figure 2B). These
findings suggest that these hub genes may lead to cabazitaxelresistance via regulation
of the immune response.
Subsequently, the focus was on identifying pathways associated with the genes related
to cabazitaxel resistance, utilizing both GO analysis and KEGG pathway analysis.
Bubble maps were constructed to illustrate the pathways exhibiting enrichment.
In GO analysis, notable enrichment was observed in the humoral immune response
pathway (Figure 2A). Simultaneously, KEGG pathway analysis revealed a
predominant enrichment in the IL-17 signaling pathway (Figure 2B). These findings
strongly imply that the hub genes may play a role in cabazitaxel resistance by
regulating the immune response.
3.3 Expression of cabazitaxel resistance-related genes in patients with PCa in
databases
3.3 Gene Expression Analysis in PCa Patients and CRPC Development

To examine whether the identified cabazitaxel resistance-related genes from CRPC

cells alter their expression upon occurrence of PCa, we analyzed the data from the
TCGA database and compared gene expression between tissues from patients with
PCa and healthy individuals. Of the 10 hub genes, only GOLGA8B was upregulated in
PCa tissues (Figure 3A). Furthermore, the expression of ITGB1, CLIP4, MAP1B,
CXCL1, C1S, and CXCL6 was downregulated upon PCa occurrence (Figure 3A). As
the TCGA database mainly includes data from the Western population, we also
analyzed the CPGEA database, which includes the data of Chinese patients with PCa.
We found that WIPI1 and GOLGA8B genes were upregulated in the Chinese patients
with PCa, while the other genes were downregulated in the patients (Figure 3B).
To investigate the potential role of cabazitaxel resistance-related genes in PCa and
CRPC, analyzed gene expression profiles using various databases the comparision f
gene expression between PCa patients and healthy individuals using the TCGA
database, with a focus on the ten identified hub genes. GOLGA8B was found to be
upregulated in PCa tissues, while ITGB1, CLIP4, MAP1B, CXCL1, C1S, and CXCL6
exhibited downregulation upon PCa occurrence (Figure 3A).To account for
population diversity because the TCGA database mainly includes data from the
Western population, extended the analysis to the CPGEA database, which includes
data from Chinese PCa patients. In this dataset, WIPI1 and GOLGA8B were
upregulated in Chinese PCa patients, while the other genes were downregulated
(Figure 3B).
Finally, we examined whether these genes show altered expression upon the
development of CRPC. For this, we analyzed another dataset, GSE35988, which
includes the data of patients with PCa and CRPC, from the GEO database. We found
that CXCL8, MMP13, and COLGA8B geneswere upregulated in the CRPC samples,
while the other genes were downregulated (Figure 3C). These findings indicate that
these cabazitaxel resistance-related genes may be important for the occurrence of both
PCa and CRPC.
Furthermore, the expression changes of these genes during CRPC development by
analyzing the GSE35988 dataset from the GEO database, which contains data from
both PCa and CRPC patients. The analysis indicated that CXCL8, MMP13, and
GOLGA8B were upregulated in CRPC samples, while the other genes exhibited
downregulation (Figure 3C). These findings collectively suggest that these
cabazitaxel resistance-related genes may play pivotal roles in the onset of both PCa
and CRPC
3.4 CXCL1andGOLGA8B affect the survival status of patients with PCa
3.4 Impact of CXCL1 and GOLGA8B on PCa Patient Survival Status

Next, we examined whether these hub genes can influence the survival status of
patients with PCa. Using the online web tool GEPIA, we analyzed the correlation
between the expression of the ten hub genes and patients’ DFS. The expression of
both CXCL1 and GOLGA8B geneswas found to be correlated with the DFS status of
patients with PCa (Figure 4).
In the following step, examination was conducted to assess the potential impact of
these hub genes on the survival outcomes of PCa patients. Utilizing the online tool
GEPIA, analysis was carried out to evaluate the correlation between the expression
levels of the ten hub genes and the disease-free survival (DFS) status of PCa patients.
Remarkably, the expression levels of CXCL1 and GOLGA8B genes exhibited
correlations with the DFS status of PCa patients (Figure 4).
3.5 GOLGA8B influences the prognosis of patients with PCa
3.5 Impact of GOLGA8B on PCa Patient Prognosis
We next confirmed whether these ten hub genes can influence the prognosis of
patients with PCa. Using the survival clinical data from the TCGA database, we
constructed single and multiple Cox regression models to determine the influence of
these genes on patient prognosis. In the single Cox regression model, CXCL1,
CXCL6, CXCL8, and GOLGA8Baffected patient prognosis (Figure 5A). However, in
the multiple Cox regression model, only GOLGA8B was identified as a risk factor
influencing patients’ prognosis (Figure 5B). Furthermore, we built a nomogram to
predict the role of these cabazitaxel resistance-related genes in affecting patients’
prognosis and found that only GOLGA8B can influence prognosis (Figure 5C).
Finally, a calibration curve was built to verify the results (Figure 5D), and this also
indicated that GOLGA8B can affect the survival and prognosis of patients with PCa.
To ascertain the potential influence of these ten hub genes on PCa patient prognosis,
survival clinical data from the TCGA database were utilized. Single and multiple Cox
regression models were constructed to assess their impact on patient prognosis. In the
single Cox regression model, CXCL1, CXCL6, CXCL8, and GOLGA8B exhibited
effects on patient prognosis (Figure 5A). However, the multiple Cox regression
model identified only GOLGA8B as a risk factor influencing patient prognosis
(Figure 5B).
Furthermore, a nomogram was developed to predict the role of these cabazitaxel
resistance-related genes in patient prognosis, revealing that GOLGA8B was the sole
gene influencing prognosis (Figure 5C). Finally, a calibration curve was constructed
to validate these results. (Figure 5D), reaffirming that GOLGA8B has the capacity to
impact the survival and prognosis of PCa patients

3.6 Correlation between GOLGA8B expression and immune cells in PCa

3.6 Association between GOLGA8B Expression and Immune Cell Infiltration in PCa
All of the above results indicated that GOLGA8Bmay be very important for PCa
development and resistance to cabazitaxel. Additionally, these cabazitaxel resistance-
related genes were correlated with immune responses. Hence, we confirmed whether
there is a correlation between GOLGA8B expressionand immune cells in PCa. Using
the data from TCGA, we analyzed the correlation between GOLGA8Bexpression
anddifferent types immune cells in PCa. GOLGA8B expression was positively
correlated with CD4+ T cells and neutrophil cells and negatively associated with B
cells, CD8+ T cells, macrophage cells, and myeloid dendritic cells (Figure 6). These
results indicated that GOLGA8B may promote PCa development and cabazitaxel
resistance by regulating immune cell infiltration.

The previous findings collectively emphasize the potential importance of GOLGA8B

in PCa development and cabazitaxel resistance. Furthermore, these genes associated
with cabazitaxel resistance have shown correlations with immune responses. As a
result, the objective was to establish a potential connection between GOLGA8B
expression and the presence of immune cells in prostate cancer (PCa). Utilizing data
from TCGA, an examination was carried out to explore the link between GOLGA8B
expression and various immune cell types within PCa. The analysis unveiled a
positive correlation between GOLGA8B expression and CD4+ T cells and neutrophil
cells, while it indicated a negative correlation with B cells, CD8+ T cells, macrophage
cells, and myeloid dendritic cells (Figure 6).These results suggest that GOLGA8B
might potentially contribute to PCa development and cabazitaxel resistance through
its influence on immune cell infiltration.
3.7 Expression of GOLGA8B in clinical PCa and CRPC samples

We also confirmed whether the expression of GOLGA8B changes upon the occurrence
of PCa in clinical patients. We collected normal para-cancer tissues and tumor tissues
and then detected the expression of GOLGA8Bat both the mRNA and protein levels.
The expression of GOLGA8B wasincreased in PCa tissues than in the normal tissues
(Figure 7A, B). Depending on the clinical data of these patients, we chose seven
paired CRPC patients and detected the expression of GOLGA8B again. We found that
GOLGA8Bwas upregulated in CRPC tissues at both the mRNA and protein levels
(Figure 7C, D). These results indicated that GOLGA8B plays a critical role in the
occurrence of both PCa and CRPC.Furthermore, confirmation was sought regarding
the changes in GOLGA8B expression associated with PCa development in clinical
patients.
In this study, researchers collected normal para-cancer tissues and tumor tissues to
assess GOLGA8B expression at both the mRNA and protein levels. The analysis
unveiled an increase in GOLGA8B expression in prostate cancer (PCa) tissues when
compared to normal tissues ( Figure 7A, B). Using clinical data from the patients
involved, seven paired castration-resistant prostate cancer (CRPC) cases were selected
for a subsequent evaluation of GOLGA8B expression. The results demonstrated an
upregulation of GOLGA8B in CRPC tissues at both the mRNA and protein levels
(Figure 7C, D). These findings underscore the pivotal role of GOLGA8B in the
development of both PCa and CRPC

3.8 Effect of GOLGA8B on cabazitaxel sensitivity of CRPC cells

3.8 Impact of GOLGA8B on Cabazitaxel Sensitivity in CRPC Cells
As GOLGA8B was upregulated in cabazitaxel-resistant CRPC cells, it may play an
important role in CRPC resistance to cabazitaxel. Hence, we examined the effect of
GOLGA8Bon the sensitivity of CRPC cells to cabazitaxel. Upon addition of
cabazitaxel to the culture medium of DU145 and PC-3 cells, the expression of
GOLGA8B increased at the mRNA and protein levels after 24 h of treatment (Figure
8A–C). Additionally, CCK-8 assay indicated that cabazitaxel can decrease the
proliferation ability of DU145 and PC-3 cells (Figure 8D, E). Then, we constructed a
GOLGA8B knock-down (shGOLGA8B) lentivirus and transfected CRPC cells with it.
We found that shGOLGA8B can decrease the expression of GOLGA8B in CRPC cells
(Figure 8F–H). CCK-8 assays further indicated that transfection of DU145 and PC-3
cells with the shGOLGA8B lentivirus decreases the proliferation ability, making the
cells more sensitive to cabazitaxel compared to the control (Figure 8I, J). These
results indicated that GOLGA8B can affect the sensitivity of CRPC cells to
cabazitaxel.
As GOLGA8B exhibited upregulation in cabazitaxel-resistant CRPC cells, it implies a
potential role in CRPC resistance to cabazitaxel.
Consequently, a study was carried out to evaluate how GOLGA8B affects the
sensitivity of CRPC cells to cabazitaxel. When cabazitaxel was introduced into the
DU145 and PC-3 cell cultures, there was a noticeable rise in the levels of GOLGA8B
expression at both the mRNA and protein levels after a 24-hour treatment period
(Figure 8A–C). Moreover, a CCK-8 assay revealed that cabazitaxel decreased the
proliferation capacity of both DU145 and PC-3 cells (Figure 8D, E)..
Subsequently, a GOLGA8B knock-down (shGOLGA8B) lentivirus was constructed
and introduced into CRPC cells. The analysis demonstrated that shGOLGA8B
effectively reduced the expression of GOLGA8B in CRPC cells (Figure 8F–H).
Further CCK-8 assays indicated that transfection of DU145 and PC-3 cells with the
shGOLGA8B lentivirus resulted in decreased proliferation ability, rendering the cells
more sensitive to cabazitaxel compared to the control (Figure 8I, J). These findings
underscore the role of GOLGA8B in modulating the sensitivity of CRPC cells to
cabazitaxel,
3.9 Effect of GOLGA8B on docetaxel sensitivity of CRPC cells
3.9 Iimpact of GOLGA8B on Docetaxel Sensitivity in CRPC Cells

As cabazitaxel is mainly used in treating docetaxel-resistant tumors, we examined the

effect of GOLGA8B on the sensitivity of CRPC cells to docetaxel. We collected
another dataset, GSE33455, which includes the RNA-sequence data of docetaxel-
sensitive and -resistant CRPC cells. We found that the docetaxel-resistant CRPC cells
showed increasing expression of GOLGA8B in comparison to the sensitive cells
(Figure 9A, B). The GSE158494 also included the data on docetaxel-resistant CRPC
cells; analysis of this dataset also revealed that docetaxel-resistant CRPC cells showed
increasing expression of GOLGA8Bcompared to the control(Figure 9C, D). Finally,
we cultured DU145 and PC-3 cells in the presence of docetaxel for 24 h, and found
that treatment with docetaxel increased the mRNA and protein expression of
GOLGA8B(Figure 9E–H). Finally, we transfected the shGOLGA8B lentivirus into
DU145 and PC-3 cells and detected the cell proliferation ability in the presence of
docetaxel. Results indicated that the knock-down of GOLGA8B renders both DU145
and PC-3 cells more sensitive to docetaxel (Figure 9I, J). Together, these results
indicated that, in addition to sensitivity tocabazitaxel, GOLGA8Bcan also influence
the sensitivity of CRPC cells to docetaxel.
In light of cabazitaxel's primary use in the treatment of docetaxel-resistant tumors, an
assessment was conducted to investigate the influence of GOLGA8B on the sensitivity
of CRPC cells to docetaxel. To achieve this, access was gained to another dataset,
GSE33455, which encompasses RNA-sequence data from docetaxel-sensitive and -
resistant CRPC cells. Analysis of this dataset revealed an upregulation of GOLGA8B
expression in docetaxel-resistant CRPC cells compared to their docetaxel-sensitive
counterparts (Figure 9A, B).The GSE158494 dataset also contained data pertaining to
docetaxel-resistant CRPC cells. Analysis of this dataset likewise unveiled an
increased expression of GOLGA8B in docetaxel-resistant CRPC cells when compared
to the control (Figure 9C, D). In another set of experiments, DU145 and PC-3 cells
were cultured in the presence of docetaxel for 24 hours, revealing that docetaxel
treatment led to an increase in the mRNA and protein expression of GOLGA8B
(Figure 9E–H). Furthermore, the shGOLGA8B lentivirus was transfected into DU145
and C-3 cells, and their proliferation ability in the presence of docetaxel was assessed.
The results demonstrated that the knock-down of GOLGA8B rendered both DU145
and PC-3 cells more sensitive to docetaxel (Figure 9I, J). Collectively, these findings
underscore that, in addition to influencing sensitivity to cabazitaxel, GOLGA8B can
also impact the sensitivity of CRPC cells to docetaxel.

4.Discussion
Owing to rapidly increasing morbidity and mortality rates, PCa has seriously
threatened the health of older men worldwide(1). Therefore, there is an urgent need
for more effective treatment to improve patients’ survival time. Many effective
methods have been used to treat PCa, with ADT being the first choice. However,
patients undergoing ADT inevitably develop CRPC, and patients entering the CRPC
stage show a median survival time of less than 20 months(18).
Due to the rapid increase in morbidity and mortality rates, prostate cancer (PCa) poses
a significant threat to the health of older men worldwide (1). Consequently, there is an
immediate requirement for more effective treatments to enhance the survival of
affected patients. Several effective approaches have been employed for PCa
treatment, with androgen deprivation therapy (ADT) being the primary option.
Nevertheless, individuals undergoing ADT ultimately develop castration-resistant
prostate cancer (CRPC), and those entering this stage typically exhibit a median
survival duration of less than 20 months (18).

Chemotherapy has been shown to be effective to treat CRPC. Chemotherapy drugs

like docetaxel and vinblastine have been used to treat CRPC and improve the survival
time of patients with CRPC(13). As an effective therapeutic drug, docetaxel can
prolong the survival time and enhance the effect of ADT in treating CRPC(19, 20).
However, patients treated with docetaxel can eventually become resistant to
docetaxel, after which intervention with other second-line chemotherapy drugs is
required.

Cabazitaxel is a type of third-generation, semisynthetic tubulin-binding taxane drug

that can be used to treat tumors resistant to other taxanes(21). In 2010, a phase III
open-label clinical trial revealed that cabazitaxel can reduce the risk of death of
patients with CRPC after the development of docetaxel resistance(11). Another study
reported that cabazitaxel can delay the progression of tumors and improve the survival
time of patients with CRPC(12). This suggests that despite many adverse effects in
the treatment of carcinomas, cabazitaxel is still an effective treatment method for
CRPC(22). Though cabazitaxel can treat docetaxel-resistant CRPC, patients may also
eventually develop resistance to cabazitaxel. Hence, finding the potential mechanisms
underlying CRPC resistance to cabazitaxel can help in better management and
treatment of CRPC. Chemotherapy has demonstrated its effectiveness in treating
CRPC. Drugs such as docetaxel and vinblastine have been employed in CRPC
treatment, leading to improved patient survival rates(13). Docetaxel, as an efficacious
therapeutic medication, can extend survival durations and enhance augment the
efficacy of ADT in CRPC treatment (19, 20).Nevertheless, individuals receiving
docetaxel treatment may eventually develop resistance, necessitating the use of
alternative second-line chemotherapy drugs. Cabazitaxel, classified as a third-
generation, semisynthetic taxane drug that binds to tubulin, is valuable for treating
tumors that have developed resistance to other taxanes (21).A phase III open-label
clinical trial in 2010 demonstrated cabazitaxel's capacity to reduce the mortality risk
in patients with CRPC following the development of docetaxel
resistance(11).Additionally, another study reported that cabazitaxel can delay tumor
progression and enhance the survival rates of CRPC patients (12).These findings
highlight cabazitaxel as an effective treatment option for CRPC, despite potential
adverse effects in carcinoma treatment (22).Nevertheless, it's important to note that
resistance to cabazitaxel can also emerge over time in patients previously responsive
to the drug. Thus, investigating the potential mechanisms underlying CRPC resistance
to cabazitaxel holds promise for improving CRPC management and treatment.

Genetic alterations have been proven to play an important role in PCa resistance to
chemotherapy drugs. Some studies had found that genetic alteration is a critical factor
for PCa resistance to chemotherapy drugs like docetaxel and vinblastine (13-15). In
addition, some studies found that genetic alterations underlie PCa resistance to
cabazitaxel. A study has reported that the upregulation of TUBB3 in PCa cells leads to
resistance to cabazitaxel(16). Another study found that the amplification of ABCB1
can cause CRPC cells to become resistant to cabazitaxel(17). These results indicate
that genetic changes are important for PCa resistance to cabazitaxel. However, until
now, there has been no systematic study on hub genes important for CRPC resistance
to cabazitaxel.
Genetic alterations have been established as pivotal contributors to PCa resistance to
chemotherapy drugs. Several studies have indicated that genetic alterations represent
a crucial factor in PCa resistance to chemotherapy agents such as docetaxel and
vinblastine (13-15).Furthermore, additional research has uncovered genetic alterations
as an underlying cause of PCa resistance to cabazitaxel. A study has reported that the
upregulation of TUBB3 in PCa cells leads to resistance to cabazitaxel(16). Another
study found that the amplification of ABCB1 can cause CRPC cells to become
resistant to cabazitaxel(17). These results indicate that genetic changes are important
for PCa resistance to cabazitaxel. However, until now, there has been no systematic
study on hub genes important for CRPC resistance to cabazitaxel
In 2020, a study used next-generation RNA-sequencing to comprehensively compare
the genes differentially expressed between cabazitaxel-sensitive and cabazitaxel-
resistant CRPC cells(23). We analyzed this reported study and identified hub genes
that may be important for CRPC resistance to cabazitaxel, and further analyzed their
function in influencing PCa progression. In our analysis, we found ten hub genes that
were upregulated in cabazitaxel-resistant CRPC cells. Further, these cabazitaxel
resistance-related genes affected the occurrence of both PCa and CRPC. A key gene
GOLGA8B was found to be able to influence the survival and prognosis of patients
with PCa. Finally, we found that GOLGA8B is upregulated in patients with PCa or
CRPC and can affect the sensitivity of CRPC cells to both cabazitaxel and docetaxel.
In 2020, a study utilized next-generation RNA-sequencing to comprehensively
compare genes with differential expression between cabazitaxel-sensitive and
cabazitaxel-resistant CRPC cells (23).Through analysis of this study, hub genes were
identified as potentially significant in CRPC resistance to cabazitaxel. Furthermore,
examination of their roles in influencing PCa progression was undertaken. Within the
analysis, upregulation of ten hub genes in cabazitaxel-resistant CRPC cells was
observed. Additionally, these genes, associated with cabazitaxel resistance,
demonstrated an impact on the development of both PCa and CRPC. Notably, a
pivotal gene, GOLGA8B, was identified as capable of influencing the survival and
prognosis of patients with PCa. Lastly, upregulation of GOLGA8B was observed in
patients with PCa or CRPC, and it was found to affect the sensitivity of CRPC cells to
both cabazitaxel and docetaxel.

GOLGA8B is associated with many diseases. Some studies have reported that changes
in GOLGA8B expression may be a reason for the occurrence of dementia and
coronary atherosclerosis(24, 25). Additionally, GOLGA8B expressionis also
correlated with the occurrence of many tumor types. A study has found that
GOLGA8B can promote lung squamous cell carcinoma by suppressing the expression
of STAT3(26). Furthermore, some long non-coding RNA can lead to the occurrence of
clear cell renal cell carcinoma by regulating the expression level of GOLGA8B(27).
Another study, which used a bioinformatics method, found that GOLGA8B may be
important for PCa(28). In this study, we found that in addition to influencing PCa
progression and prognosis, GOLGA8B is an important gene for CRPC resistance to
both cabazitaxel and docetaxel.
GOLGA8B is associated with various diseases. Studies have suggested that alterations
in GOLGA8B expression may contribute to the development of conditions such as
dementia and coronary atherosclerosis (24, 25).Additionally, GOLGA8B expression
has been linked to the occurrence of multiple types of tumors. For instance, one study
revealed that GOLGA8B can promote lung squamous cell carcinoma by suppressing
the expression of STAT3(26). Furthermore, certain long non-coding RNAs have a role
in the development of renal cell carcinoma by regulating GOLGA8B expression
levels (27).Another study, utilizing bioinformatics approaches, highlighted the
potential significance of GOLGA8B in PCa (28).Within this study, it was observed that
GOLGA8B not only influences PCa progression and prognosis but also serves as a
critical gene in conferring resistance to both cabazitaxel and docetaxel in CRPC
However, our study has some limitations. First, the cabazitaxel-resistant CRPC data
were from cell lines and not clinical samples. Data derived from the cell lines alone
may differ greatly from clinical data. But, as clinical cabazitaxel-resistant CRPC
samples are very hard to collect, we could not analyze clinical data. Hence, the
identified cabazitaxel resistance-related genes may not be comprehensive and the
occurrence of bias is a possibility. Second, though we tested the expression of
GOLGA8B in clinical PCa and CRPC samples, the sample size was small. Hence, the
obtained results may not be truly representative, and a larger sample size will be
needed to validate the results. Third, though we found that GOLGA8Bis a crucial gene
for CRPC resistance to cabazitaxel, the underlying mechanism remains unclear. Thus,
in the future, studies should focus on uncovering the mechanism by which GOLGA8B
promotes CRPC resistance to cabazitaxel.
Nevertheless, this study does possess several limitations. Firstly, the data regarding
cabazitaxel-resistant CRPC were obtained from cell lines rather than clinical samples.
The data from cell lines alone may substantially differ from clinical data. However,
the collection of clinical cabazitaxel-resistant CRPC samples is extremely
challenging, rendering our analysis limited to cell lines. Consequently, there is a
possibility of incompleteness in identifying cabazitaxel resistance-related genes, and
bias could be present. Secondly, while assessing GOLGA8B expression in clinical
PCa and CRPC samples, the sample size was small. Therefore, the findings may not
be fully representative, and the results will require validation with a larger sample
size.
Thirdly, although ascertained that GOLGA8B plays a pivotal role in CRPC resistance
to cabazitaxel, the precise underlying mechanism remains unclear. Consequently,
future research should prioritize uncovering the mechanism through which GOLGA8B
promotes CRPC resistance to cabazitaxel
5.Conclusion
In conclusion, we found ten hub genes that may play important roles in CRPC
resistance to cabazitaxel. Furthermore, we showed that GOLGA8B plays a pivotal role
in CRPC occurrence and development and may be a key regulator of CRPC resistance
to cabazitaxel.
In conclusion, ten hub genes were identified that may play important roles in
cabazitaxel resistance in CRPC. Furthermore, it was demonstrated GOLGA8B that
plays a pivotal role in the occurrence and development of CRPC and may serve as a
key regulator of cabazitaxel resistance in CRPC.