C-terminal kinesin motor KIFC1 participates in facilitating proper cell division of human seminoma

Abstract

C-terminus kinesin motor KIFC1 is known for centrosome clustering in cancer cells with supernumerary centrosomes. KIFC1 crosslinks and glides on microtubules (MT) to assist normal bipolar spindle formation to avoid multi-polar cell division, which might be fatal. Testis cancer is the most common human cancer among young men. However, the gene expression profiles of testis cancer is still not complete and the expression of the C-terminus kinesin motor KIFC1 in testis cancer has not yet been examined. We found that KIFC1 is enriched in seminoma tissues in both mRNA level and protein level, and is specifically enriched in the cells that divide actively. Cell experiments showed that KIFC1 may be essential in cell division, but not essential in metastasis. Based on subcellular immuno-florescent staining results, we also described the localization of KIFC1 during cell cycle. By expressing ΔC-FLAG peptide in the cells, we found that the tail domain of KIFC1 might be essential for the dynamic disassociation of KIFC1, and the motor domain of KIFC1 might be essential for the degradation of KIFC1. Our work provides a new perspective for seminoma research.

Keywords: KIFC1; cell division; kinesin-14; seminoma; testis cancer.

Figure 1. KIFC1 is enriched in human seminoma samples in both mRNA and protein level

(A) Comparison of mRNA expression level among human muscle (mus), testis (tes) and seminoma (sem) tissue samples. kifc1 mRNA is enriched in seminoma samples. (B) Comparison of protein expression level among human muscle (mus), testis (tes) and seminoma (sem) tissue samples. KIFC1 protein is enriched in both testis samples and seminoma samples, and KIFC1 protein expression level of seminoma samples is significantly higher than that of testis samples. (C-J) HE staining results of human muscle tissues (C, D) and testis tissues (E, F) near the seminoma tissue and the seminoma tissue (G, H) from seminoma patient. (I) The transition of the seminoma to the muscle tissue. (J) The invasion of the cancer cells into the testis tissue. Black arrows show infiltrating lymphocytes. Blue arrows show seminiferous tubules of normal testis tissue. Red arrows show cancer cells. Yellow arrow shows muscle tissue. In the both testis and seminoma, a number of lymphocyte infiltration with bleeding are showed. In seminoma tissues, compared with normal tissues, seminiferous tubules were disrupted and replaced by seminoma cells with loose nuclei and watery cytoplasm.

Figure 2. Localization of KIFC1 in seminoma tissue samples and nearby testis tissue samples from human determined by immuno-florescent staining

KIFC1 is omnipresent in both seminoma samples and testis samples and co-localizes with MT. White arrows show seminoma cells with loose nuclei and watery cytoplasm, in which KIFC1 signal is strong. Yellow arrows show other cells such as lymphocytes, in which KIFC1 signal is weak. Compared with normal testis tissue, in seminoma tissues the characteristic structure of seminiferous tubules is completely disrupted, and KIFC1 has a tendency to be concentrated around the loose nuclei.

Figure 3. Knock down of KIFC1 in HeLa cells by RNAi

(A) WB analysis of knock down efficiencies using three different sets of siRNA. (B) Localization of KIFC1 in HeLa cells determined by immuno-florescent staining. (C) Quantification of WB band intensity from (A). The knock down efficiency is as follows: KD2 > KD1 > KD3. (D) Growth curve of cells with KIFC1 knock down. The negative control is transfected with siRNA targeting GFP. A=A450-A650, and A0 represents the A value at the time point of 0. (E-X) Dynamic behavior of cells with different levels of KIFC1 knock down was determined by wound-healing assay.

Figure 4. Localization of KIFC1 during cell cycle in HeLa cells determined by immuno-florescent staining

(A-H) KIFC1 is rapidly transported into and concentrated in the nucleus upon synthesized. In metaphase, KIFC1 is mainly localized on the spindle, but some of it also spread in the cytoplasm. Later on, KIFC1 is degraded after mitotic exit. (I) A model illustrating the localization of KIFC1 during the cell cycle.

Figure 5. FLAG-tagged ΔC in HeLa cells

(A) FLAG-tagged ΔC can be expressed in HeLa cells. (B) Localization of pCMV-N-FLAG plasmid (control) and FLAG-tagged ΔC during cell cycle in HeLa cells determined by immuno-florescent staining. (C) Growth curve of cells transfected with FLAG-tagged ΔC or transfected pCMV-N-FLAG plasmid (control). (D) Dynamic behavior of cells transfected with FLAG-tagged ΔC or transfected pCMV-N-FLAG plasmid (control) determined by wound-healing assay. (E-K) Endogenous KIFC1 will be entering the nucleus and finally be concentrated in the nucleus during prophase.