Kinectin 1 promotes the growth of triple-negative breast cancer via directly co-activating NF-kappaB/p65 and enhancing its transcriptional activity

Triple-negative breast cancer (TNBC) is the most challenging subtype of breast cancer. Various endeavor has been made to explore the molecular biology basis of TNBC. Herein, we reported a novel function of factor Kinectin 1 (KTN1) as a carcinogenic promoter in TNBC. KTN1 expression in TNBC was increased compared with adjacent tissues or luminal or Her2 subtypes of breast cancer, and TNBC patients with high KTN1 expression have poor prognosis. In functional studies, knockdown of KTN1 inhibited the proliferation and invasiveness of TNBC both in vitro and in vivo, while overexpression of KTN1 promoted cancer cell proliferation and invasiveness. RNA-seq analysis revealed that the interaction of cytokine-cytokine receptor, particularly CXCL8 gene, was upregulated by KTN1, which was supported by the further experiments. CXCL8 depletion inhibited the tumorigenesis and progression of TNBC. Additionally, rescue experiments validated that KTN1-mediated cell growth acceleration in TNBC was dependent on CXCL8 both in vitro and in vivo. Furthermore, it was found that KTN1 enhanced the phosphorylation of NF-κB/p65 protein at Ser536 site, and specifically bound to NF-κB/p65 protein in the nucleus and cytoplasm of cells. Moreover, the transcription of CXCL8 gene was directly upregulated by the complex of KTN1 and NF-κB/p65 protein. Taken together, our results elucidated a novel mechanism of KTN1 gene in TNBC tumorigenesis and progression. KTN1 may be a potential molecular target for the development of TNBC treatment.


Ethics statement
The obtained BC tissue arrays was supported by the National Human Genetic
All cell lines used in this study were authenticated using short tandem repeat typing (results are presented in the Supplementary data 2) and tested to exclude mycoplasma contamination.

Cell Proliferation and Colony Formation Assay
For cell proliferation assay, MDA-MB-231(8000 per well) or BT549 cells (6000 per well) cultivated on 96-well plates (Corning) were transfected with siRNA oligos, and cell proliferation were determined after 0h, 24h, 48h, and 72h by CCK-8 kit (MCE) at 450nm according to the manufacturers' instructions. For cell colony formation assay, MDA-MB-231(1000 per well) or BT549 cells (1000 per well) were cultivated on 6-well plates. Attached cells were transfected with siRNA oligos, and cells were collected at 7 day. Cells were washed twice with PBS and fixed in 4% paraformaldehyde for 1h. Cell colonies were photographed and counted after staining with 0.1% crystal violet.

Cell Migration and Invasiveness
For cell migration assay treated with KTN1 shRNA vectors, KTN1 overexpression with minicircle KTN1 vectors, and CXCL8 siRNA oligos, 1×10 5 cells were seeded in Transwell chambers (Merck Millipore, 8µm) of 24-welll plates (Corning). The complete medium (10% FBS) was added to the bottom of Transwell chambers, while the serum-free medium was added to the top of Transwell chambers. After being cultured between 16h and 20h, the cells were fixed with 4% paraformaldehyde, photographed, and calculated for statistical analysis. Each experiment was repeated at least three times.
For cell migration assay and cell invasion assay treated with KTN1 siRNA oligos or CXCL8 protein, 1×10 5 cells were seeded in Transwell chambers (Merck Millipore, 8µm) of 24-welll plates (Corning), respectively. The complete medium (5% FBS) containing 800 ng/ml CXCL8 protein was added to the bottom of Transwell chambers, while the serum-free medium containing cells was added to the top of Transwell chambers. Other measures were the same as the above contents.

RNA extraction, Quantitative Reverse Transcription-PCR (qRT-PCR), and RNA sequencing
Total RNA was extracted from cells using TRIzol (Life Technologies) according to the manufacturer's instructions. Reverse transcription was conducted using TransScript One-Step RT-PCR SuperMix (Transgen Biotech), and qPCRs were run using SYBR Green PCR Master Mix (Thermo Fisher Scientific) in a in 96-well plate (Bio-Rad). The mRNA relative expression was calculated by the comparative Ct values. Each experiment was repeated at least three times. The primer sequences for qRT-PCR were listed in Table S1. For RNA sequencing, NEB library were prepared to obtain mRNA, subjected to quality control using an Agilent 2100 bioanalyzer. RNA sequencing was performed at Beijing Novogene, using the Illumina Novaseq 6000 platform. Table S1. Sequences of qRT-PCR primers used in this study.  of cases were showed below. Data were plotted as the means of 95% confidence interval ± s.d. One-Way ANOVA and Dunnett's multiple comparison test were used to analyze the data. *P <0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 µm.