Identification of DYNLT1 associated with proliferation, relapse, and metastasis in breast cancer

Background Breast cancer (BC) is the most common malignant disease worldwide. Although the survival rate is improved in recent years, the prognosis is still bleak once recurrence and metastasis occur. It is vital to investigate more efficient biomarkers for predicting the metastasis and relapse of BC. DYNLT1 has been reported that participating in the progression of multiple cancers. However, there is still a lack of study about the correlation between DYNLT1 and BC. Methods In this study, we evaluated and validated the expression pattern and prognostic implication of DYNLT1 in BC with multiple public cohorts and BC tumor microarrays (TMAs) of paraffin-embedded tissues collected from the Affiliated Hospital of Jining Medical University. The response biomarkers for immune therapy, such as tumor mutational burden (TMB), between different DYNLT1 expression level BC samples were investigated using data from the TCGA-BRCA cohort utilizing public online tools. In addition, colony formation and transwell assay were conducted to verify the effects of DYNLT1 in BC cell line proliferation and invasion. Results The results demonstrated that DYNLT1 overexpressed in BC and predicted poor relapse-free survival in our own BC TMA cohort. In addition, DYNLT1 induced BC development by promoting MDA-MB-231 cell proliferation migration, and metastasis. Conclusion Altogether, our findings proposed that DYNLT1 could be a diagnostic and prognostic indicator in BC.


. Introduction
Breast carcinoma (BC) is the most common cancer in women and ranks the second leading cause of tumor-related death in women in the United States and in China (1,2).According to the molecular pathological types, there are various therapeutic strategies for BC, such as surgical therapy, radiation therapy, chemotherapy, endocrine therapy, and targeted therapy (3).The response to treatment and prognosis of BC relies on molecular characteristics that have been well established, and the molecular type based on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) status shows excellent performance for guiding clinicians to select the optimal treatment for BC patients in the past few decades (4,5).In addition, with the development of modern genomic and transcriptomic technologies, numerous gene markers are identified for predicting the response to treatment and prognosis of cancer (6,7).The prognosis of BC is improved in the past few decades; however, there are still numerous women dying from BC, especially triple-negative breast cancer (TNBC), in the world.As a result, it is urgent to explore a more specific molecular target to direct the diagnosis and treatment of BC.
In this study, we identified that dynein light chain tctex type 1 (DYNLT1), a component of the cytoplasmic dynein 1 complex, may predict the prognosis of BC.DYNLT1 is responsible for the intracellular retrograde motility of vesicles and organelles along microtubules, binding to transport cargo, and is involved in apical cargo transport.It is reported that DYNLT1 plays an important role in many biological functions and diseases, such as Huntington's disease (8), fertilizing potential of human spermatozoa (9), migration of epidermal cells in hypoxia (10), autophagy lysosomal degradation (11), and several types of cancer.DYNLT1 has been reported to promote glioblastoma progression and is associated with tumor-node-metastasis (TNM) grade (12).In gastric cancer (GC), DYNLT1 takes part in the miR-15b-3p/Caspase-3/Caspase-9 signaling pathway to promote malignant transformation (13).However, it is still unclear whether DYNLT1 is related to BC.
In this study, we evaluated the mRNA expression of DYNLT1 between BC and normal breast tissues from multiple public cohorts and validated the results at the protein level by immunohistochemistry (IHC) staining for DYNLT1 in 68 BC samples along with paired 55 adjacent normal breast specimens collected from the Affiliated Hospital of Jining Medical University.In addition, we observed that DYNLT1 affected the migratory and colony-forming abilities of BC cells in vitro.Furthermore, in vivo experiment was conducted to verify that DYNLT1 knockdown suppressed tumor growth and abolished distant metastasis.Therefore, we proposed that DYNLT1 may have the potential to become a promising diagnostic indicator and prognostic predictor of BC patients.

. . GEPIA . database
The GEPIA 2.0 (http://gepia2.cancer-pku.cn/#index)database (18) was utilized to evaluate the mRNA expression of DYNLT1 in pan cancers using data from The Cancer Genome Atlas (TCGA).In addition, GEPIA 2.0 was also utilized to evaluate the prognostic implication of DYNLT1 in pan cancers.

. . Breast cancer gene-expression miner database
The Breast Cancer Gene-Expression Miner (http://bcgenex.centregauducheau.fr/BC-GEM/GEM-Accueil.php?js=1) database (19) was utilized to evaluate the mRNA expression of DYNLT1 in subgroups of BC samples stratified based on multiple clinicpathological features.

. . GO and KEGG analyses
DYNLT1 and its potential interacting proteins were utilized to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by R software with the "clusterProfiler" package (22).Terms with a false discovery rate (FDR) of <0.05 were illustrated.

. . Human BC specimens
A total of 68 BC samples paired with 55 normal breast tissues collected from the Affiliated Hospital of Jining Medical University were approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University (approval number: 2021-08-C015).All participants provided written informed consent.

. . Colony-forming assay
MDA-MB-231 cells were seeded at a density of 200 cells per well in six-well plates.Single cells were cultured in DMEM with 10% FBS at 37 • C with 5% CO 2 for 3 weeks.Colonies were fixed with 10% formalin and then stained with 0.1% crystal violet.

. . Cell growth assay
Lentivirus-infected stable cells were seeded into 96well plates and cultured in 10% FBS DMEM (2,000 cells per well, five parallel wells).Then, the cells were collected at different points in time, and the cell number in each well was counted by the CCK-8 reagent.The absorbance at 450 nm was employed to determine the number of viable cells.

. . Transwell assay
The migration assays and invasion assays were performed using a transwell chamber (Corning).A total of 2 × 10 4 cells per well were seeded into the upper chamber for the migration assays, while 5 × 10 4 cells per well were seeded into the upper chamber after matrigel was coagulated at 37 • C for the invasion assays with serum-free medium, and the bottom of the chamber contained the DMEM medium with 10% FBS.Cells were fixed by 10% formalin and stained by 0.1% crystal violet after migration for 24 h.Migrated BC cells' pictures were captured by an inverted light microscope at ×100 magnification, and three random fields were counted.

. . Statistical analysis
Student's t-test and chi-square test were utilized to analyze the difference between the two groups.A P-value of <0.05 was considered statistically significant.

. . DYNLT expression was higher in BC compared to normal breast tissues
First, we evaluated the mRNA expression of DYNLT1 across 33 cancer types and paired normal samples with data from TCGA by the GEPIA database.Our results demonstrated that the mRNA expression of DYNLT1 was higher in most types of cancer tissues compared with paired normal samples, such as BC, GBM, LGG, and PAAD (Figures 1A,  B).Next, four GEO cohorts were utilized to validate the result that the mRNA expression of DYNLT1 was higher in BC tissues compared to paired normal breast samples (Figures 1C-F).In addition, subgroup analysis of multiple clinic pathological features of BC samples in the Breast Cancer Gene-Expression Miner database showed that DYNLT1 expression is related to HER-2 status, PAM50-based intrinsic subtype, Scarff-Bloom-Richardson (SBR) grade, Nottingham prognostic index (NPI), and age and mutation status of TP53 (Figures 2A-F). . .High expression of DYNLT predicting poor overall relapse and distant metastasis-free survival of BC First, we evaluated the prognostic implications of DYNLT1 in 33 cancer types using data from TCGA by the GEPIA database.Our results demonstrated that DYNLT1 was a significant (P < 0.05) risk factor for the prognosis of BC, MESO, and LIHC (Figures 3A, B).Furthermore, we validated the prognostic implication of DYNLT1 in BC by the PrgnoScan database.Our results identified that high expression of DYNLT1 predicted poor disease-specific survival (Figures 3C, D), relapse-free survival (Figures 3E-H), and distant metastasis-free survival (Figure 3I) in BC.

. . Validated the expression pattern and prognostic implication of BC by TMA
To explore the correlation of DYNLT1 and the characteristic of BC patients at the protein level, a BC TMA, containing 68 cancer tissues were used for IHC staining for DYNLT1.The results validated that DYNLT1 expressed significantly higher in BC tissues than in adjacent normal breast samples (Figures 4A, B).High expression of DYNLT1 was positively correlated with higher TNM stage (Figure 4C) and predicted poor relapse-free survival (RFS) with a log-rank P-value of <0.015 (Figure 4D).

. . PPI network and functional annotation of DYNLT
We constructed a PPI network combining DYNLT1 and its potential interacting proteins by the String database.The result indicated that DYNLT1 may be capable of interacting with BMPR2, DYNC1H1, DYNC1I1, DYNC1I2, DYNC1LI1, DYNC1LI2, DYNLL1, DYNLL2, DYNLRB1, and WDR34 (Figure 5A).The gene ontology and pathway functional enrichment analyses of genes in this network demonstrated that these genes may involve Frontiers in Medicine frontiersin.org in cell cycle, cell division, immune system process, vesiclemediated transport, motor activity, protein binding, phagosome, adaptive immune system, and apoptosis (Figures 5B-D).
. .DYNLT as a predictive biomarker for immune checkpoint blocking therapy in patients with BC Generally, a high DNA damage repair (DDR) mutational ratio, a high proportion of TMB, high neoantigen loads, high TILs regional fraction, and low TGF-beta response predict well overcome in ICB therapy for patients with cancer (31, 32).In our study, the results of GSEA analysis based on data from TCGA-BRCA showed that DNA replication (Figure 6A) pathways were enriched in DYNLT1 high BC samples.In addition, we found that DTNLT1 expression was positively related to DNA damage and DNA repair (Pearson's correlation > 0.3, P < 0.05) at the single cell level of BC by the CancerSEA database (Figure 6B).We found that BC patients in the DYNLT1 high group had higher levels of DDR mutational ratio (Figure 6C), TILs regional fraction (Figure 6D), and lower TGF-beta response (Figure 6E) compared to those in the DYNLT1 low group.Moreover, a high proportion of neoantigen loads (Figure 6F) and TMB (Figure 6G) was shown in the DYNLT1 high group, and the CIBERSORT analysis showed that BC samples in the DYNLT1 high group had a higher infiltration ratio of CD8+ T cells and follicular helper T cells than those in the DYNLT1 low group (Figure 6H).

. . DYNLT knockdown suppressed the colony-forming, proliferation, migratory, and invasion abilities of BC cells
To explore the functional role of DYNLT1 in BC cells, in vitro experiments were conducted.First, MDA-MB-231-shDYNLT1 and MDA-MB-231-sh scramble (NC) stable cell lines were constructed successfully by lentiviral transduction (Figure 7A).Next, the results of the colony-forming assay and CCK-8 assays demonstrated that knockdown expression of DYNLT1 inhibited the colony formation and proliferation abilities in MDA-MB-231 cells (Figures 7B, C).Furthermore, a transwell assay was conducted to confirm that migration and invasion abilities were also attenuated with the DYNLT1 knockdown in MDA-MB-231 cells (Figures 7D, E).

. . Knockdown of DYNLT suppressed tumor growth and abolished the lung and liver metastasis in vivo
To determine the role of DYNLT1 in vivo, MDA-MB-231-shDYNLT1 and MDA-MB-231-shNC cells were injected into a mammary fat pad in severe combined immunodeficiency (SCID) female mice.Knockdown of DYNLT1 led to smaller tumor volume (Figures 8A, B) and poor survival (Figure 8C).The lung and the liver samples of sacrificed mice were formalin-fixed and paraffinembedded, and HE stain was conducted to evaluate the distant metastasis.As expected, the knockdown of DYNLT1 prevented tumor cells from metastasizing to distant organs including the lung and the liver, which prolong the survival period of tumor-burden mice (Figures 8D, E).

. Discussion
With the development of modern genomic/transcriptomic technologies and increasing public cancer genomic programs, such  as TCGA and GEO, numerous biomarkers were identified for improving our ability to diagnose and treat cancer or utilized as predictors of prognosis and response to therapies in cancer.For example, DYNLT1 serving as a prognostic indicator for GBM patients has been reported (12).However, there is no report on whether DYNLT1 may act as a biomarker of BC.
In our study, we validated that DYNLT1 expression was higher in BC than normal breast tissues by integrated bioinformatics analysis using data from multiple public cohorts and ourselves BC and adjacent normal breast specimens.In addition, we demonstrated that high DYNLT1 expression meant a poor prognosis in BC, and DYNLT1 knockdown suppressed MDA-MB-231 cell migration and colony formation.Furthermore, in in vivo experiment, we found that DYNLT1 knockdown suppressed tumor growth and abolished distant metastasis.Mice with DYNLT1 knockdown tumor cells survived a longer period.However, there is still a limitation in our study.The molecular mechanism of how high DYNLT1 expression enhances BC cells' proliferative and invasive abilities remains unclear.
Abnormal proliferative and invasive abilities are the leading causes of progression and poor prognosis in BC.It has been reported that DYNLT1 promoted migration, invasion, and proliferation, and inhibited apoptosis of GC via the exo-miR-15b-3p/DYNLT1/Caspase-3/Caspase-9 pathway (13).Previous studies also found that DYNLT1 interacted with the tumor suppressor REIC/Dkk-3 which induced malignant cell death via modification of the Wnt signaling pathway (33, 34).In addition, Kawasaki et al. (35) demonstrated that REIC/Dkk-3 overexpression could induce multidrug-resistant BC cell line MCF-7 apoptosis via downregulating P-glycoprotein.Hence, a high expression of DYNLT1 can promote the progression of BC via interacting with REIC/Dkk-3.
Recent evidence indicates that a high proportion of TMB and loads of neoantigens predicted a good response for ICB therapy in many types of cancer (36, 37), and patients with high TILs and low TGF-beta response in tumor samples always carry a better prognostic significance in ICB treatment (38,39).Our results showed that biomarkers representing an effective response to ICB treatment were always accompanied by DYNLT1 high expression in BC samples.Therefore, we inferred that DYNLT1 may be a response biomarker in ICB therapy for BC.

. Conclusion
Our study first suggested that DYNLT1 may serve as a biomarker for diagnosing and ICB treating BC or a predictor for predicting the prognosis of BC.

FIGURE
FIGURE DYNLT mRNA expression was higher in BC tissues compared to normal breast tissues.(A) DYNLT mRNA expression across cancer types (TCGA).(B) DYNLT mRNA expression between BC tissues and normal breast tissues in the TCGA-BRCA cohort.(C) DYNLT mRNA expression between BC tissues and normal breast tissues in the GSE cohort.(D) DYNLT mRNA expression between BC tissues and normal breast tissues in the GSE cohort.(E) DYNLT mRNA expression between BC tissues and normal breast tissues in the GSE cohort.(F) DYNLT mRNA expression between BC tissues and normal breast tissues in the GSE cohort.TCGA, The Cancer Genome Atlas; *P < ., ***P < ., and ****P < ..

FIGURE
FIGURE Subtype analysis of DYNLT mRNA expression based on clinic-pathological features of BC tissues.(A) Subtype analysis of DYNLT mRNA expression based on HER status.(B) Subtype analysis of DYNLT mRNA expression based on PAM subtype.(C) Subtype analysis of DYNLT mRNA expression based on SBR grade.(D) Subtype analysis of DYNLT mRNA expression based on NPI.(E) Subtype analysis of DYNLT mRNA expression based on age.(F) Subtype analysis of DYNLT mRNA expression based on TP mutant status.HER-, human epidermal growth factor receptor-; SBR, Scar -Bloom-Richardson; NPI, Nottingham prognostic index.

Female
FIGURE High DYNLT expression predicted poor survival in BC. (A) High expression predicted poor prognosis in BC, LGG, and LIHC in the GEPIA database.(B) High DYNLT expression predicted poor overall survival (OS) of BC patients in the TCGA-BRCA cohort.(C, D) High DYNLT expression predicted poor disease-specific survival (DSS) of BC patients in the GSE cohort (C) and GSE cohort (D).(E-H) High DYNLT expression predicted poor relapse-free survival (RFS) of BC patients in the GSE cohort (E), GSE cohort (F), GSE cohort (G), and GSE cohort (H).(I) High DYNLT expression predicted poor distant metastasis-free survival (DMFS) of BC patients in the GSE cohort.

FIGURE
FIGURE Validated the protein expression pattern and prognostic implication of DYNLT in BC. (A) Protein expression of DYNLT was higher in BC compared to normal breast tissues.(B) Scatter plot of TMED IHC score between BC and normal breast tissues.(C) Scatter plot of TMED IHC score between TNM stage I BC and TNM stage II+III BC tissues.(D) Kaplan-Meier analysis was utilized to compare the relapse-free survival between high DYNLT expression BC patients and low DYNLT expression BC patients.IHC, immunohistochemistry. ***P < .and ****P < . .

FIGURE
FIGURE DYNLT may predict well overcome in immune checkpoint blocked therapy for patients with BC. (A) GSEA analysis showed that the DNA replication pathway was enriched in DYNLT High BC samples.(B) DTNLT mRNA expression was positively related to DNA damage, DNA repair, apoptosis, hypoxia, TILs, and invasion at the single BC cell level.(C) The DDR pathways' mutational ratio between DYNLT High and DYNLT Low BC samples.(D) The regional fraction between DYNLT High and DYNLT Low BC samples.(E) The TGF-beta response score between DYNLT High and DYNLT Low BC samples.(F) The neoantigen loads between DYNLT High and DYNLT Low BC samples.(G) The TMB between DYNLT High and DYNLT Low BC samples.(H) The infiltration ratio of immune cells between DYNLT High and DYNLT Low BC samples.TMB, tumor mutational burden; DDR, DNA damage repair; TILs, tumor-infiltrating lymphocytes; BER, base excision repair; NER, nucleotide excision repair; MMR, mismatch repair; FA, Fanconi anemia; HR, homologous recombination; NHEJ, non-homologous end joining; DR, direct repair; TLS, translesion synthesis; *P < .; **P < .; ***P < .; and ****P < . .

FIGURE
FIGURE DYNLT knockdown inhibited MDA-MB-cells' colony formation and migration.(A) Knockdown expression of DYNLT in MDA-MB-cells.(B) DYNLT knockdown inhibited cell colony formation and histogram of colony numbers between the DYNLT knockdown group and control group.(C) CCK-assay of the DYNLT knockdown group and control group.(D) DYNLT knockdown inhibited cell migration and histogram of cell counts between the DYNLT knockdown group and control group.(E) DYNLT knockdown inhibited cell invasion and histogram of cell counts between the DYNLT knockdown group and the control group.Scale bar = µm, ***P < ., **P < . .

FIGURE
FIGURE DYNLT knockdown suppressed tumor growth and abolished lung and liver metastasis in vivo.(A) DYNLT knockdown in MDA-MB-cells suppresses tumor growth in SCID mice.(B) Tumor samples were shown after mice were sacrificed.(C) Survival of SCID mice with MDA-MB--shNC tumor or MDA-MB--shDYNLT # tumor.(D) Representative lung and liver HE staining of mice with MDA-MB--shNC tumor or MDA-MB--shDYNLT # tumor (scale bar = mm).(E) The number of tumor metastatic sites in the lung and the liver (n = biological replicates).*P < ., **P < ., and ****P < ..