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KIAA1114, a full-length protein encoded by the trophinin gene, is a novel surface marker for isolating tumor-initiating cells of multiple hepatocellular carcinoma subtypes.

Kim SW, Yang HG, Kang MC, Lee S, Namkoong H, Lee SW, Sung YC - Oncotarget (2014)

Bottom Line: Notably, KIAA1114 expression was strongly detected in primary hepatic tumor, but neither in the adjacent non-tumorous tissue from the same patient nor normal liver tissue.KIAA1114high cells isolated from HCC cell lines displayed TIC-like features with superior functional and phenotypic traits compared to their KIAA1114low counterparts, including tumorigenic abilities in xenotransplantation model, in vitro colony- and spheroid-forming capabilities, expression of stemness-associated genes, and migratory capacity.Our findings not only address the value of a novel antigen, KIAA1114, as a potential diagnostic factor of human liver cancer, but also as an independent biomarker for identifying TIC populations that could be broadly applied to the heterogeneous HCC subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyungbuk, Republic of Korea.

ABSTRACT
Identification of novel biomarkers for tumor-initiating cells (TICs) is of critical importance for developing diagnostic and therapeutic strategies against cancers. Here we identified the role of KIAA1114, a full-length translational product of the trophinin gene, as a distinctive marker for TICs in human liver cancer by developing a DNA vaccine-induced monoclonal antibody targeting the putative extracellular domain of KIAA1114. Compared with other established markers of liver TICs, KIAA1114 was unique in that its expression was detected in both alpha fetoprotein (AFP)-positive and AFP-negative hepatocellular carcinoma (HCC) cell lines with the expression levels of KIAA1114 being positively correlated to their tumorigenic potentials. Notably, KIAA1114 expression was strongly detected in primary hepatic tumor, but neither in the adjacent non-tumorous tissue from the same patient nor normal liver tissue. KIAA1114high cells isolated from HCC cell lines displayed TIC-like features with superior functional and phenotypic traits compared to their KIAA1114low counterparts, including tumorigenic abilities in xenotransplantation model, in vitro colony- and spheroid-forming capabilities, expression of stemness-associated genes, and migratory capacity. Our findings not only address the value of a novel antigen, KIAA1114, as a potential diagnostic factor of human liver cancer, but also as an independent biomarker for identifying TIC populations that could be broadly applied to the heterogeneous HCC subtypes.

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TIC-like properties of KIAA1114high HCC cells(A) Flow cytometric analysis of sorted KIAA1114high and KIAA1114low cells from HuH7 and SK-Hep-1 cell lines. (B) Colony formation assay of KIAA1114high and KIAA1114low fractions isolated from HuH7 and SK-Hep-1 cells. 2,000 sorted cells were seeded into 6-well plates in triplicate and cultured for 2 weeks. The resulting colonies were stained with crystal violet and counted under the microscope. Representative images are shown. *p <0.05; **p<0.01. (C) Sphere forming assay of purified cells. 2,000 KIAA1114high and KIAA1114low cells were plated in 6-well ultra-low attachment dishes and cultured for 2 weeks. Spheres were counted and photographed using the microscope. Scale bars: 500 μm. **p <0.01. (D) Quantitative real-time PCR analysis of stemness-related genes in KIAA1114high and KIAA1114low cells derived from HuH7 and SK-Hep-1.*p <0.05; **p<0.01. (E) Transwell migration assay of KIAA1114high and KIAA1114low subsets. 5 × 104 HuH7-sorted or 1 × 104 SK-Hep-1-sorted cells were seeded onto the upper chamber and incubated for 16 hours. Migrated cells were stained with Diff-Quik and counted. Representative microscopic fields are shown. Scale bars: 500 μm. **p <0.01. (F) Representative images of beige/nude/xid mice subcutaneously injected with 1 × 104 HuH7-sorted or 1 × 105 SK-Hep-1-sorted KIAA1114high cells (black arrow) and KIAA1114low cells (white arrow). The photographs were taken 3 months after injections. All graphically represented data are shown as average ± SEM.
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Figure 4: TIC-like properties of KIAA1114high HCC cells(A) Flow cytometric analysis of sorted KIAA1114high and KIAA1114low cells from HuH7 and SK-Hep-1 cell lines. (B) Colony formation assay of KIAA1114high and KIAA1114low fractions isolated from HuH7 and SK-Hep-1 cells. 2,000 sorted cells were seeded into 6-well plates in triplicate and cultured for 2 weeks. The resulting colonies were stained with crystal violet and counted under the microscope. Representative images are shown. *p <0.05; **p<0.01. (C) Sphere forming assay of purified cells. 2,000 KIAA1114high and KIAA1114low cells were plated in 6-well ultra-low attachment dishes and cultured for 2 weeks. Spheres were counted and photographed using the microscope. Scale bars: 500 μm. **p <0.01. (D) Quantitative real-time PCR analysis of stemness-related genes in KIAA1114high and KIAA1114low cells derived from HuH7 and SK-Hep-1.*p <0.05; **p<0.01. (E) Transwell migration assay of KIAA1114high and KIAA1114low subsets. 5 × 104 HuH7-sorted or 1 × 104 SK-Hep-1-sorted cells were seeded onto the upper chamber and incubated for 16 hours. Migrated cells were stained with Diff-Quik and counted. Representative microscopic fields are shown. Scale bars: 500 μm. **p <0.01. (F) Representative images of beige/nude/xid mice subcutaneously injected with 1 × 104 HuH7-sorted or 1 × 105 SK-Hep-1-sorted KIAA1114high cells (black arrow) and KIAA1114low cells (white arrow). The photographs were taken 3 months after injections. All graphically represented data are shown as average ± SEM.

Mentions: Nextly, to investigate whether KIAA1114 could play a role as an independent marker to identify liver TICs, we isolated cell populations expressing high and low levels of KIAA1114 from AFP+ HuH7 cells and AFP− SK-Hep-1 cells and comparatively assessed their functional capacities of TICs. As with previous studies of CD133 and EpCAM expression analysis in human liver cancer [8, 26], HCC cell lines examined in this study exhibited a Gaussian distribution of KIAA1114 expression levels, rather than displaying a discrete, isolated population of KIAA1114+ cells. Therefore, sorted fractions obtained by FACS-selecting the top 10% most brightly stained cells were designated “KIAA1114high” cells, whereas those derived from the bottom 10% most dimly stained cells were designated “KIAA1114low” cells. Assuming 95% purities of KIAA1114low cells after separation, the purities of KIAA1114high cells in HuH7 and SK-Hep-1 cells were 84.3% and 57.6%, respectively (Figure 4A). Nextly, to evaluate clonogenic potential of isolated subpopulations, colony formation assay was performed. The result showed that the number of colonies generated from KIAA1114high populations was greater than that from KIAA1114low counterparts, regardless of AFP expression status of parental cells (Figure 4B). Analogously, sphere-formation assay, which was used as readout for self-renewal activity, revealed that KIAA1114high cells isolated from either HuH7 or SK-Hep-1 cell line formed significantly more spheroids than did KIAA1114low cells (Figure 4C). These findings suggest that KIAA1114high subsets possess higher proliferative potential and self-renewal capacity than KIAA1114low counterparts. We also noticed differences in qualitative aspects of spheres formed by AFP+ HuH7 and AFP− SK-Hep-1 cells. The size of each spheroid generated by KIAA1114high fraction was much larger than that of KIAA1114low fraction-derived one in HuH7 cells, while the spheres formed by KIAA1114high and KIAA1114low cells sorted from SK-Hep-1 cells were similar in size. Moreover, the overall sphere-forming efficiencies of sorted cells were relatively lower in SK-Hep-1 cells compared to HuH7 cells.


KIAA1114, a full-length protein encoded by the trophinin gene, is a novel surface marker for isolating tumor-initiating cells of multiple hepatocellular carcinoma subtypes.

Kim SW, Yang HG, Kang MC, Lee S, Namkoong H, Lee SW, Sung YC - Oncotarget (2014)

TIC-like properties of KIAA1114high HCC cells(A) Flow cytometric analysis of sorted KIAA1114high and KIAA1114low cells from HuH7 and SK-Hep-1 cell lines. (B) Colony formation assay of KIAA1114high and KIAA1114low fractions isolated from HuH7 and SK-Hep-1 cells. 2,000 sorted cells were seeded into 6-well plates in triplicate and cultured for 2 weeks. The resulting colonies were stained with crystal violet and counted under the microscope. Representative images are shown. *p <0.05; **p<0.01. (C) Sphere forming assay of purified cells. 2,000 KIAA1114high and KIAA1114low cells were plated in 6-well ultra-low attachment dishes and cultured for 2 weeks. Spheres were counted and photographed using the microscope. Scale bars: 500 μm. **p <0.01. (D) Quantitative real-time PCR analysis of stemness-related genes in KIAA1114high and KIAA1114low cells derived from HuH7 and SK-Hep-1.*p <0.05; **p<0.01. (E) Transwell migration assay of KIAA1114high and KIAA1114low subsets. 5 × 104 HuH7-sorted or 1 × 104 SK-Hep-1-sorted cells were seeded onto the upper chamber and incubated for 16 hours. Migrated cells were stained with Diff-Quik and counted. Representative microscopic fields are shown. Scale bars: 500 μm. **p <0.01. (F) Representative images of beige/nude/xid mice subcutaneously injected with 1 × 104 HuH7-sorted or 1 × 105 SK-Hep-1-sorted KIAA1114high cells (black arrow) and KIAA1114low cells (white arrow). The photographs were taken 3 months after injections. All graphically represented data are shown as average ± SEM.
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Figure 4: TIC-like properties of KIAA1114high HCC cells(A) Flow cytometric analysis of sorted KIAA1114high and KIAA1114low cells from HuH7 and SK-Hep-1 cell lines. (B) Colony formation assay of KIAA1114high and KIAA1114low fractions isolated from HuH7 and SK-Hep-1 cells. 2,000 sorted cells were seeded into 6-well plates in triplicate and cultured for 2 weeks. The resulting colonies were stained with crystal violet and counted under the microscope. Representative images are shown. *p <0.05; **p<0.01. (C) Sphere forming assay of purified cells. 2,000 KIAA1114high and KIAA1114low cells were plated in 6-well ultra-low attachment dishes and cultured for 2 weeks. Spheres were counted and photographed using the microscope. Scale bars: 500 μm. **p <0.01. (D) Quantitative real-time PCR analysis of stemness-related genes in KIAA1114high and KIAA1114low cells derived from HuH7 and SK-Hep-1.*p <0.05; **p<0.01. (E) Transwell migration assay of KIAA1114high and KIAA1114low subsets. 5 × 104 HuH7-sorted or 1 × 104 SK-Hep-1-sorted cells were seeded onto the upper chamber and incubated for 16 hours. Migrated cells were stained with Diff-Quik and counted. Representative microscopic fields are shown. Scale bars: 500 μm. **p <0.01. (F) Representative images of beige/nude/xid mice subcutaneously injected with 1 × 104 HuH7-sorted or 1 × 105 SK-Hep-1-sorted KIAA1114high cells (black arrow) and KIAA1114low cells (white arrow). The photographs were taken 3 months after injections. All graphically represented data are shown as average ± SEM.
Mentions: Nextly, to investigate whether KIAA1114 could play a role as an independent marker to identify liver TICs, we isolated cell populations expressing high and low levels of KIAA1114 from AFP+ HuH7 cells and AFP− SK-Hep-1 cells and comparatively assessed their functional capacities of TICs. As with previous studies of CD133 and EpCAM expression analysis in human liver cancer [8, 26], HCC cell lines examined in this study exhibited a Gaussian distribution of KIAA1114 expression levels, rather than displaying a discrete, isolated population of KIAA1114+ cells. Therefore, sorted fractions obtained by FACS-selecting the top 10% most brightly stained cells were designated “KIAA1114high” cells, whereas those derived from the bottom 10% most dimly stained cells were designated “KIAA1114low” cells. Assuming 95% purities of KIAA1114low cells after separation, the purities of KIAA1114high cells in HuH7 and SK-Hep-1 cells were 84.3% and 57.6%, respectively (Figure 4A). Nextly, to evaluate clonogenic potential of isolated subpopulations, colony formation assay was performed. The result showed that the number of colonies generated from KIAA1114high populations was greater than that from KIAA1114low counterparts, regardless of AFP expression status of parental cells (Figure 4B). Analogously, sphere-formation assay, which was used as readout for self-renewal activity, revealed that KIAA1114high cells isolated from either HuH7 or SK-Hep-1 cell line formed significantly more spheroids than did KIAA1114low cells (Figure 4C). These findings suggest that KIAA1114high subsets possess higher proliferative potential and self-renewal capacity than KIAA1114low counterparts. We also noticed differences in qualitative aspects of spheres formed by AFP+ HuH7 and AFP− SK-Hep-1 cells. The size of each spheroid generated by KIAA1114high fraction was much larger than that of KIAA1114low fraction-derived one in HuH7 cells, while the spheres formed by KIAA1114high and KIAA1114low cells sorted from SK-Hep-1 cells were similar in size. Moreover, the overall sphere-forming efficiencies of sorted cells were relatively lower in SK-Hep-1 cells compared to HuH7 cells.

Bottom Line: Notably, KIAA1114 expression was strongly detected in primary hepatic tumor, but neither in the adjacent non-tumorous tissue from the same patient nor normal liver tissue.KIAA1114high cells isolated from HCC cell lines displayed TIC-like features with superior functional and phenotypic traits compared to their KIAA1114low counterparts, including tumorigenic abilities in xenotransplantation model, in vitro colony- and spheroid-forming capabilities, expression of stemness-associated genes, and migratory capacity.Our findings not only address the value of a novel antigen, KIAA1114, as a potential diagnostic factor of human liver cancer, but also as an independent biomarker for identifying TIC populations that could be broadly applied to the heterogeneous HCC subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyungbuk, Republic of Korea.

ABSTRACT
Identification of novel biomarkers for tumor-initiating cells (TICs) is of critical importance for developing diagnostic and therapeutic strategies against cancers. Here we identified the role of KIAA1114, a full-length translational product of the trophinin gene, as a distinctive marker for TICs in human liver cancer by developing a DNA vaccine-induced monoclonal antibody targeting the putative extracellular domain of KIAA1114. Compared with other established markers of liver TICs, KIAA1114 was unique in that its expression was detected in both alpha fetoprotein (AFP)-positive and AFP-negative hepatocellular carcinoma (HCC) cell lines with the expression levels of KIAA1114 being positively correlated to their tumorigenic potentials. Notably, KIAA1114 expression was strongly detected in primary hepatic tumor, but neither in the adjacent non-tumorous tissue from the same patient nor normal liver tissue. KIAA1114high cells isolated from HCC cell lines displayed TIC-like features with superior functional and phenotypic traits compared to their KIAA1114low counterparts, including tumorigenic abilities in xenotransplantation model, in vitro colony- and spheroid-forming capabilities, expression of stemness-associated genes, and migratory capacity. Our findings not only address the value of a novel antigen, KIAA1114, as a potential diagnostic factor of human liver cancer, but also as an independent biomarker for identifying TIC populations that could be broadly applied to the heterogeneous HCC subtypes.

Show MeSH
Related in: MedlinePlus