HIF-1α affects invasion and metastasis of prostate cancer by regulating invadopodia formation

doi:10.21203/rs.3.rs-4867481/v1

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Research Article

HIF-1α affects invasion and metastasis of prostate cancer by regulating invadopodia formation

https://doi.org/10.21203/rs.3.rs-4867481/v1

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Background

Prostate cancer is an important disease that threatens the health of middle-aged and older men. In tumor diseases, HIF-1α can affect tumor proliferation, invasion, and metastasis by regulating the expression of various types of molecules and thus activating related signaling pathways. In this study, we explored the mechanisms by which HIF-1α affects prostate cancer invasion and metastasis.

Method

Exploration of the mechanisms by which HIF-1α affects prostate cancer invasion and metastasis using transcriptome sequencing. Detection of invadopodia-related molecules using Western blot. The formation of invadopodia in prostate cancer cells and the localization of HIF-1α and TKS5 were detected using immunofluorescence. Detection of extracellular matrix degradation by invadopodia in prostate cancer using gelatin degradation assays. Validation of the interaction of HIF-1α with TKS5 using Co-IP.

Results

HIF-1α is highly expressed in prostate cancer. HIF-1α can affect the proliferation, invasion, and metastasis of prostate cancer. We found that HIF-1α was associated with cell motility as well as extracellular matrix degradation in prostate cancer, suggesting that HIF-1α may promote prostate cancer invasion and metastasis by affecting the formation of invadopodia. Degradation of extracellular matrix by prostate cancer cells was reduced after knockdown of HIF-1α, as was the formation of invadopodia. The results of Western blot showed that the expression of TKS5 and MMP9 was decreased after knockdown of HIF-1α. The results of Co-IP and immunofluorescence suggested that HIF-1α could interact with TKS5 near the nuclear membrane.

Conclusion

In the present study, we demonstrated that HIF-1α was able to influence the invasive and metastatic ability of prostate cells by affecting the expression of TKS5 and thus the formation of invadopodia. We also found that HIF-1α was able to interact with TKS5 near the nuclear membrane. These results provide a new direction for the exploration of invadopodia in the prostate cancer.

prostate cancer

hypoxia-inducible factor 1α

invadopodia

invasion

metastasis

Hypoxia as a common pathophysiologic state is seen in a variety of diseases.[1] One of the characteristics of tumors is hypoxia.[2] In tumor diseases, although tumors are able to secrete relevant factors to promote vascular growth, the distribution of these neovascularization is disordered and endothelial discontinuity, so that neovascularization does not contribute to oxygen delivery, and at the same time, tumors are chronically hypermetabolic, and the consumption of oxygen is higher than that of normal tissues, therefore, the hypoxic microenvironment of tumors is caused by the inability of abnormal blood vessels to provide sufficient oxygen for the high metabolic state of tumor cells.[3–5] In the state of hypoxia, tumor cells tend to exhibit characteristic properties. Tumor cell proliferation, invasion and migration are enhanced by hypoxia, while the transcription and translation of relevant genes can be caused by hypoxia and thus the regulation of downstream pathways.[6, 7]

For tumor cells that are in the absence of oxygen, the activation of hypoxia-inducible factors is the main reason why tumors are able to survive in hypoxic environments, and it is also an important molecule that influences the biological behavior of tumor cells in hypoxic environments.[8, 9] The HIF family contains two subunits, the members with the α-subunit are HIF-1α, HIF-2α, and HIF-3α, and the member with the β-subunit is HIF-3β.[10, 11] HIF-2α and HIF-1α are tumor initiating factors, of which HIF-1α is broadly distributed in vivo, whereas HIF-2α is abundantly distributed in the placenta, heart, lungs, and endothelial cells of adult blood vessels, and HIF-3α, which has a function opposite to that of the former two, is thought to function to inhibit the actions of the former two.[12–15] In the HIF family HIF-1α is the most characteristic isoform and plays a key role in driving tumor progression.[16, 17] With enough oxygen, HIF-1α undergoes hydroxylation by oxygen-dependent prolyl hydroxylase and degradation occurs via E3 ligase binding to VHL tumor suppressor proteins and reacting with each other; however, in hypoxic environments, the activity of oxygen-dependent prolyl hydroxylase is inhibited and the degradation of HIF-1α is reduced, which leads to an abnormal build-up of HIF-1α in the cell.[18, 19] HIF-1α has two transcription activation domain structure, N-TAD and C-TAD, C-TAD regulation of HIF-1α transcriptional activity, while N-TAD mainly regulates the oxygen stability of HIF-1α.[20, 21] Two transcription factors can be enhanced by HIF-1α in the state of hypoxia, Twist and Snail, which attenuate the expression of E-calreticulin and thus promote epithelial mesenchymal transition, which occurs when tumor cells lose polarity to gain higher invasiveness.[22, 23] Mutated oncogenes such as p53 and PTEN can stimulate the HIF-1α system to promote tumor angiogenesis.[24] HIF-1α also affects polymerase expression thereby regulating collagen fiber alignment and integrin activity, which in turn promotes tumor metastasis.[25]

A class of outwardly convex structures on the tumor cell membrane that are in contact with the ECM and have the ability to degrade it are known as invadopodia.[26] The invadopodia allow for enhanced motor migration of tumor cells, providing the impetus for tumor metastasis.[27] The composition of the invadopodia is complex, and proteomics studies have shown that the components of the invadopodia are involved in many cellular functions.[28] Mature invadopodia are converted from invadopdium “precursors”, which need to be anchored to the plasma membrane by TKS5 regulation.[29, 30] In previous studies, invadopodia have been reported in prostate cancer,[31] and the association of HIF-1α with invadopodia has not been investigated. We verified the association of HIF-1α with invadopodia and confirmed that HIF-1α increased the transcription of TKS5 thereby promoting the invadopodia formation in prostate cancer cells. In addition, we found that HIF-1α was able to interact with TKS5.

Patients and tissue samples: benign prostatic hyperplasia tissues and prostate cancer tissue during 2016 to 2021 were obtained from the Department of Urology, The First Affiliated Hospital of Chongqing Medical University (Chongqing, China). The characteristics of the included patients are shown in TableⅠ. Immunohistochemistry public data from The Human Protein Atlas (https://www.proteinatlas.org/).

Cell lines and transfection: PC-3, DU145, LNCaP, RWPE-1 and 22RV1 used in this study were gifted by the Molecular Medicine and Oncology Research Center, Chongqing Medical University, Chongqing, China. PC-3 cells were cultured in Ham's F-12K medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. DU145 was cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin. LNCaP, 22RV1 and RWPE-1 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin. All cell lines were placed in the incubator at 37°, 5% CO₂. All cell culture procedures were performed under mycoplasma-free conditions. Lentiviruses and plasmids for knockdown and overexpression of HIF-1α were purchased from Genechem (Shanghai, China) as well as Sangon Biotech (Shanghai, China), respectively, and were transfected according to the protocols provided by the manufacturers. The shRNA sequences were as follows: shRNA for HIF-1α(shHIF-1α), 5’-AATGTGAGTTCGCATCTTGAT-3’; shRNA for negative control (shNC), 5’-TTCTCCGAACGTGTCACGT-3’.

Wound-healing: Cells were inoculated into six-well plates and scratched with a 200uL pipette tip when the healing rate reached 100%, and samples were taken and photographed at 0 hour and 24 hours.

Trans-well: Cells were cultured in serum-free medium for 24 hours and then transferred to Trans-well chambers (LANSELECT, Anhui, China). 24 hours later, the chambers were removed and fixed by adding 4% paraformaldehyde, and then stained by adding drops of crystal violet.

Cell Counting Kit-8: PC3 cells were inoculated into 96-well plates according to 100 µl of culture solution per well, and assayed at 0h, 24h, 48h and 72h using CCK-8 kit (Beyotime, C0038). Each well was incubated at 37°C for 1h by adding 10µl CCK-8 solution, and the absorbance was measured at 450nm.

Cell cycle assay: Cells in six-well plates were collected and washed once with PBS, then 1 mL of DNA Staining solution and 10 µl of Permeabilization solution were added and mixed well, and incubated at room temperature and protected from light for 30 minutes, and finally detected using flow cytometry (CytoFLEX, Beckman).

Gelatin degradation assay: Configure the fluorescent gelatin solution according to the instructions. 100 µl of fluorescent gelatin solution was added dropwise to the confocal dish, incubated at room temperature for 10 min and then washed twice with PBS, fixed using 4% paraformaldehyde at room temperature, washed three times with PBS after fixation was completed, and 400 µl of complete medium was added and incubated for 4 hours at 37°C. 400uL of cell suspension was added dropwise to the fluorescent gelatin crawler, incubated at 37°, 5% CO2 incubator for 8 hours and then removed from the 24-well plate, fixed using 4% paraformaldehyde, and the follow-up same as immunofluorescence.

Animal experiments: Male BALB/c-nu thymus-free nude mice (4 weeks old, 20-25g, 20 in total, subcutaneous tumor models = 10, bone metastasis models = 10) were divided into two groups (shNC group, n = 5 and shHIF-1α group, n = 5). shNC PC-3 cells or shHIF-1α PC-3 cells were injected into the bone marrow cavities of the tibiae of the mice as well as subcutaneously, and the tumorigenic effect was monitored one week after injection.

Immunohistochemistry: Paraffin sections were placed in a 60° oven for 2 hours, then the sections were immersed in xylene for 45 minutes, and then sequentially placed in graded ethanol solutions (100%; 95%; 85%; 75%) for 5 minutes per gradient. Antigenic repair of the tissues was performed using sodium citrate solution, and the tissues were processed for staining using an immunohistochemistry kit (ZSGB-BIO, Beijing, China) after the repair was completed. In the assays against TKS5, HIF-1α, MMP9, and Ki67, we used primary antibodies including HIF-1α (1:200, Abcam, ab51608), TKS5 (1:200, Proteintech, 18976-1-AP), MMP9 (1:200, Proteintech, 30592-1-AP), Ki67 (1:200, Proteintech, 28074-1-AP). The average absorbance of the tissues was measured using Image J.

Immunofluorescence: Cells were inoculated into confocal dishes and cultured for 3 h. After the cells were fully attached to the wall, the medium was discarded and the residual medium was washed away with PBS, 4% paraformaldehyde was added and fixed for 10 min, and then goat serum was added for blocking, and after blocking was completed, the primary antibody was added and incubated overnight. In the assays against TKS5, F-actin, and HIF-1α, we used primary antibodies including TKS5 (1:200, Proteintech, 18976-1-AP), Phalloidin-AF555 (1:200, ZEN BIO, 16002), HIF-1α (1:200, Abcam. ab51608). Secondary antibody with HRP labeling was added and then fluorescent dye was added for staining. After staining was completed, DAPI was added to stain the nuclei, and finally an anti-fluorescence quencher was added dropwise. Cells were observed and photographed using a laser confocal microscope (Leica TCS SP8).

Coi

mmunoprecipitation (CoIP): Samples were processed to obtain precipitates using the Pierce™ Classic Magnetic IP/Co IP Kit(Thermo Fisher Scientific, 88804) according to the instruction manual, and the precipitates were denatured and assayed using western blot.

Western blot: Protein samples were obtained by processing the tissues/cells using the protein extraction kit(Beyotime, Shanghai, China), and after addition of 5×SDS buffer༈Beyotime, Shanghai, China༉, the samples were boiled at 95 ° C for 10 minutes and stored at 4 ° C. Use 6% and 8% SDS-PAGE gel for electrophoretic separation of proteins, after the completion of electrophoresis, the proteins on the gel will be electrotransferred to the PVDF membrane, and then use the skim milk powder solution configured with TBST for blocking, after the completion of blocking, wash off the excess block solution and put it into a primary antibody solution for incubation. After incubation, the excess primary antibody solution was washed away and incubated in the secondary antibody solution, and finally the ECL (Abbkine, BMU102) was configured for developing. The primary and secondary antibodies we used included TKS5 (1:1000, Proteintech, 18976-1-AP), HIF-1α (1:1000, Proteintech, 20960-1-AP), MMP9 (1:1000, Proteintech, 10375-2-AP), GAPDH (1: 5000, ZEN BIO, R24404), Goat Anti-Rabbit IgG (1:10000, biosharp, BL003A). Bands were analyzed for grayscale values using Image J.

Bioinformatic analysis: Use correlation analysis of the GEPIA database(http://gepia.cancer-pku.cn/) to detect the association of HIF-1α with invadopodia-related molecules.

Transcriptome sequencing: RNA was extracted using Total RNA Extractor (Trizol) (Sangon Biotech, B511311) and libraries were created. Raw data were obtained using Illumina Hiseq and analysed for differences using DESeq. Differential genes were annotated using GO and KEGG. Functionally enriched correlation analysis using the R’s igraph package.

Liquid Chromatography-Mass Spectrometry (LC-MS): Thermo Scientific LC-MS system (Thermo Fisher Scientific) was used to analyze the potential interaction sites between HIF-1α and TKS5.

Quantitative reverse transcription PCR, RT-qPCR: Total RNA was extracted by RNA extraction kit (RK30120, ABclonal), and the concentration of RNA was detected by NanoDrop2000(Thermo Fisher Scientific). Follow the instructions. cDNA was obtained using reverse transcription kit (RK20429, ABclonal). The reaction conditions are as follows: 37℃for 2min,55℃ for 15min ,85℃ for 5min. qPCR was performed using the CFX Connect Real-Time System(Bio-Rad),and use the following protocol: Denaturation at 95°C for 3 min, followed by 95°C for 5 sec, 60°C for 30 sec, 40 cycles. Using the 2ΔΔCq to analysis the relative gene expression levels. The sequences were as follows: HIF-1α: F: 5’-GGCGCGAACGACAAGAAAAA-3’ ,R: 5’-GCCGAGATCTGGCTGCAT-3’;GAPDH: F༚5’-CAGGAGGCATTGCTGATGAT-3’, R: 5’-GAAGGCTGGGGCTCATTT-3’.

Statistical analysis: GraphPad (version 10.1.2, GraphPad Software Inc.) and SPSS (version 27.0.1, IBM Corp.) were used for plotting and statistics. Comparisons between two groups were performed using an unpaired Student's ttest, comparisons between multiple groups were performed using oneway ANOVA followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.

1. Expression of HIF-1α in prostate cancer

In our previous study, the results of protein microarray suggested that HIF-1α showed high expression in PCa. We also validated the expression of HIF-1α in BPH as well as prostate cancer tissues using PCR(Fig.1C). According to the degree of invasive infiltration of the tumor, pathologically prostate cancer is classified as T1 to T4. The prognosis is more optimistic for patients with early stages prostate cancer, but for stages as T3 or T4, the tumor will break through the peripheral membrane and grow. To preliminarily explore whether HIF-1α is associated with prostate cancer aggressiveness, T1-T4 prostate cancer specimens and benign prostatic hyperplasia specimens were examined for HIF-1α expression, and the results of immunohistochemistry showed that the expression of HIF-1α increased with the progression of prostate cancer stage, which suggested that the high expression of HIF-1α was associated with prostate cancer aggressiveness (Fig.1A and B). Immunohistochemistry based on public databases showed a trend towards increased expression of HIF-1α in high-grade prostate cancer (Fig.1D). Based on the above evidence we hypothesized that HIF-1α can affect the capacity of prostate cancer cells to invade.

2. HIF-1α affects the invasion and migration of prostate cancer cells

In the cell experiments, we selected the PC-3 cell line which has a higher level of HIF-1α expression than other cell lines (Fig. 2A and B), and also the PC3 cell line has a higher invasive and metastatic ability as cells derived from bone metastatic prostate cancer. For assessing the effect of HIF-1α on prostate cancer cell invasion and migration, we performed Trans-well and wound-healing on shNC cells as well as shHIF-1α cell, and the results showed that knockdown of HIF-1α resulted in fewer invasive cells and a reduced rate of migration as compared to the shNC group (Fig.2C-F). We also performed rescue experiments on PC-3 with knockdown of HIF-1α. After overexpression against HIF-1α, the Invasive and migratory capacity of PC-3 cells were restored (Fig.2C-F). We also verified the effect of HIF-1α on the cell cycl, after knockdown of HIF-1α, the number of cells in the dividing phase was reduced, and similarly, the number of cells in the dividing phase was increased after rescue experiments targeting HIF-1α (Fig.2G and H). Similar trends were also found in the CCK-8 assay (Fig.2I). Bone metastases occur well in the advanced stages of prostate cancer,[32] and bone metastases from prostate cancer are often indicative of a severely poor prognosis. To verify the effect of HIF-1α on prostate cancer in vivo, we established a subcutaneous tumorigenic model as well as a bone metastasis model in nude mice, and found that the growth efficiency of PC-3 cells was lower than that of the control group after knockdown of HIF-1α (Fig.3D), the weight and volume of the tumors in nude mice were lower in shHIF-1α group (Fig.3A and C); at the same time, the degree of damage to the bone quality of the femur in nude mice caused by PC-3 was reduced after knockdown of HIF-1α (Fig.3B). In the detection of Ki67 in tumor specimens, the number of Ki67-positive cells in the experimental group was lower than that in the control group (Fig.3E), which indicated that HIF-1α can affect prostate cancer proliferation in vivo environment. Collectively, these results suggest that in vivo and in vitro environments prostate cancer invasion and metastasis are affected by HIF-1α.

3. HIF-1α promotes tumor invasion and metastasis by affecting cell motility

HIF-1α as a transcription factor activates downstream gene expression. In order to investigate in what way HIF-1α promotes prostate cancer invasion and metastasis, we sequenced the transcriptome of the PC-3 cells, and we found that a total of 1180 genes were down-regulated and 975 genes were up-regulated after down-regulation of HIF-1α in PC3 cells (Fig.4A). GO enrichment analysis showed that the up-regulation of HIF-1α was closely related to the cell motility (Fig.4D), and KEGG enrichment analysis revealed that high expression of HIF-1α was able to associate with relevant pathways regulating cell motility, as well as interacting with extracellular matrix receptors (Fig.4C, E). Therefore, we hypothesized that HIF-1α may be able to promote invasion and metastasis of prostate cancer cells by affecting cell motility and ECM degradation. Previous studies have found that cell motility and invadopodia formation are linked. The formation of invadopodia is important for tumor cell metastasis, and invadopodia are specialized structures that come into contact with and are capable of degrading the ECM. The transcriptome sequencing results also revealed that the movement of cellular components correlates with MMP9 (Fig.4B), which is one component of invadopodia to initiate ECM degradation,[33] which on the other hand supports our hypothesis that HIF-1α can promote the formation of invadopodia in prostate cancer cells. To further test whether the expression of HIF-1α correlates with the invadopodia-related molecules, we analyzed these molecules using public databases, and we found that HIF-1α positive correlates with TKS5, Cortactin, Cofilin1, MMP-9 (Fig.4F), which act on the formation of invadopodium “precursor”, stabilization of invadopodium “precursor”, and maturation of invadopodia.

4. HIF-1α affects ECM degradation in PC3 cells by regulating invadopodia formation

To assess the effect of HIF-1α on the formation of invadopodia, we examined the co-location of TKS5 and F-actin[34] after knockdown of HIF-1α, and found that knockdown of HIF-1α reduces the formation of invadopodia in PC-3 cells. (Fig.5B and C). Also knockdown of HIF-1α inhibited gelatin degradation due to invadopodia (Fig5A and C). In rescue experiments, HIF-1α increased the formation of invadopodia and the degradation of gelatin by invadopodia(Fig.5A-C). Mature invadopodia are attached to the cell membrane, usually located below the nucleus.[35] Previous studies have shown that invadopodium “precursors” are formed first during the formation of invadopodia, and the anchoring of invadopodium “precursors” to the cell membrane is mainly regulated by tyrosine kinase 5 (TKS5)[30]. We used the Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze prostate cancer tissues and results showed that the phosphorylation site of TKS5 is closely related to HIF-1α (Data S1); therefore, in the following study, we explored the relationship between HIF-1α and TKS5. After knocking down the expression of HIF-1α, we found that the downregulation of TKS5 and MMP9 also occurred, and when HIF-1α got upregulated in the rescue experiment, the upregulation of TKS5 and MMP9 also happened (Fig.5D and E). In the in vivo experiments, immunohistochemical assays against TKS5 and MMP9 showed the same results (Fig.5F), which suggests that HIF-1α may affect the subsequent development of invadopodia by influencing TKS5. Meanwhile, in order to explore whether HIF-1α and TKS5 can interact, we co-localized HIF-1α as well as TKS5 using immunofluorescence, and the results showed that they co-localized at the nuclear membran, which suggested that HIF-1α might be able to interact with TKS5, and the results of the Co-IP suggested the existence of HIF-1α-TKS5 interaction (Fig.5G and H). Overall, in prostate cancer cells, HIF-1α can affect the formation of invadopodia by affecting TKS5, this promotes the ability of PC3 cells to invade, migrate.

From our study, HIF-1α can promote PC3 cell invasion and migration by regulating the invadopodia formation and the effect can be explained at two levels; HIF-1α affects invadopodia formation by promoting the expression of TKS5; and HIF-1α can interact with TKS5 in the vicinity of the nuclear membrane of the cell. (Fig. 6)

The incidence and mortality rates of prostate cancer in worldwide show a rising trend year by year, and prostate cancer has now become a major disease that threatens the health of men.[36, 37] Among the causes of death in prostate cancer patients, metastasis of cancer is an important factor, and bone metastasis occurs well in clinical. Tumors are often found in a hypoxic environment and tumor development promotes a hypoxic environment.[38] We examined the expression of HIF-1α in prostate cancer as well as in BPH, and we found that HIF-1α was higher in prostate cancer. After analyzing the patients in combination with their pathological stages, we found that the expression level of HIF-1α was related to the stage of prostate cancer. Therefore, we speculated that the high expression of HIF-1α promoted the invasion and migration of prostate cancer, and the cellular experiments and animal experiments also preliminarily confirmed this view. HIF-1α belongs to the transcription factors, so we knocked down HIF-1α in PC-3 cells and completed transcriptome sequencing, and found that HIF-1α was associated with cell motility as well as extracellular matrix degradation, so we hypothesized that HIF-1α may be involved in cellular motility by affecting invadopodia formation and thus promoting PC-3 cell invasion and metastasis. Meanwhile, we tested the correlation of HIF-1α and key molecules of invadopodia using public databases, and found that HIF-1α correlated with TKS5, Cortactin, Cofilin1, MMP-9, however, it is noteworthy that we did not find Cortactin in the differential gene analysis based on transcriptome sequencing, suggesting that the effect of HIF-1α on Cortactin may not exist at the transcriptional level but at the protein modification level. The stabilization and anchoring of invasive pseudopod precursors are regulated by TKS5, and protein profiling suggests that TKS5 is affected by hypoxia, so we chose to explore the relationship between HIF-1α and TKS5. Hypoxia is one of the factors promoting the formation of invadopodia, and in neck and head, pancreatic, and lung cancer cells HIF-1α can promote the formation of invadopodia by up-regulating the level of HB-EGF[39], but through which way affects the formation of invadopodia in prostate cancer cells has not been described in detail. After knocking down HIF-1α, the expression of TKS5 and MMP9 also consistently underwent a decrease, suggesting that HIF-1α is able to influence the expression level of TKS5 and thus the formation of invadopodia. Similarly, in animal experiments, the expression of TKS5 and MMP9 was reduced in the experimental group. We designed a gelatin degradation assay to explore whether HIF-1α has an effect on extracellular matrix degradation, and we also performed fluorescence co-staining of invadopodia, and found that the formation of invadopodia was reduced after knockdown of HIF-1α, and the area of gelatin degradation was reduced. The above experiments suggest that HIF-1α is indeed able to influence the formation of invadopodia and subsequent degradation of the extracellular matrix by impressing TKS5. However, in our previous work we also found that TKS5 and HIF-1α co-localization near the nucleus, suggesting that they might interact with each other near the nucleus, in order to validate this idea, we designed CO-IP to verify whether the two could interact with each other, and the results showed that HIF-1α and TKS5 were able to interact with each other. Taken together, the effect of HIF-1α on the formation of invadopodia may be carried out at two different levels: HIF-1α promotes the transcription of TKS5; and HIF-1α has protein interactions with TKS5 near the nuclear membrane of the nucleus. However, the formation of invadopodia is a complex process, and whether HIF-1α can interact with other molecules is something that needs to continue to be explored. In terms of TKS5 itself, TKS5 is a protein composed of multiple substructures, and it has been reported that the activity of TKS5 is regulated by its own substructures.[40, 41] Whether HIF-1α is able to affect the activity of TKS5 by binding to TKS5 fragments is a follow-up of the present study; similarly, in terms of the transcription of TKS5, in what way HIF-1α up-regulates TKS5 expression will also be a follow-up of our study.

AOD, average optical density; BPH, benign prostatic hyperplasia; CCK-8, cell counting Kit-8; Co-IP, co immunoprecipitation; Cortactin, cortical actin-binding protein; ECL, Enhanced chemiluminescence; ECM, extracellular matrix; GO, gene ontology; HIF, hypoxia-inducing factors; KEGG, kyoto encyclopedia of genes and genomes; LC-MS, liquid chromatography-mass spectrometry; MMP9, matrix metalloprotein9; PCa, prostate cancer; TKS5, tyrosine kinase5.

Acknowledgement：

Not applicable.

Funding：

This work was supported by the following two grants: 1. Grants from doctoral program of the first affiliated hospital of Chongqing Medical University (CYYY-BSYJSCXXM-202332); 2. Technology innovation and application development of Chongqing Science and Technology Commission (CSTB2023TIAD-KPX0053)

Availability of data and materials:

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions:

Shuai Su and Liangdong Song designed the research program. Liangdong Song, Shuai Su, Jindong Zhang and Huixuan Zhu performed the experiments and analyzed the data. Delin Wang provided resources. Yu Luo, Chengcheng Wei, Kun Han and Yunfan Li collected specimens and patient characteristics.

Ethics approval and consent to participate:

This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Approval No.2022-K234). In this study, each patient's privacy is protected. The animal study was approved by the Experimental Animal Ethics Committee of Chongqing Medical University.

Patient consent for publication:

Not applicable.

Competing interests:

The authors declare that they have no competing interests.

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TableⅠ.Characteristics of the included patients

Group	Number of patients	HIF-1α expression Level （AOD: Mean±SD）	P value	t/F value
Age（years）
＜60	35	24.86±10.28	0.5411	t=0.6158
≥60	12	22.73±10.66	0.5411	t=0.6158
BMI	47	24.84±2.19	NA	NA
Tumor size
T1	4	7.04±0.85	<0.001	F=395.0
T2	20	17.86±1.57
T3	13	27.30±2.53
T4	10	40.27±2.21
Lymph node metastasis
N0	43	24.01±9.96	0.8822	t=0.1490
N1	4	23.58±13.40	0.8822	t=0.1490
Metastasis
M0	41	23.59±9.69	0.2076	t=1.279
M1	6	29.30±13.65	0.2076	t=1.279
Gleason Score
Grade 1: ≤6	5	25.83±11.70	0.4081	F=1.020
Grade 2: 3+4=7	8	26.85±12.64
Grade 3: 4+3=7	12	26.08±11.02
Grade 4: 4+4=8 5+3=8 3+5=8	7	17.96±6.78
Grade 5: ≥9	15	22.98±7.84

No competing interests reported.

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HIF-1α affects invasion and metastasis of prostate cancer by regulating invadopodia formation

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