Xenotransplantation elicits salient tumorigenicity of adult T-cell leukemia-derived cells via aberrant AKT activation

Abstract

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells.

Keywords: Adult T-cell leukemia; proto-oncogene protein Akt; severe combined immunodeficient mice; tumor-initiating cells; xenotransplantation.

Figure 1

Increased tumorigenicity of adult T‐cell leukemia/lymphoma‐derived cell lines by serial transplantation. (a) Tumor growth of ST1 and ST1‐N6 cells in NOG mice. After cell injection (10⁴ cells/site), the sizes of the developed tumors were measured by a caliper and tumor volumes were plotted. To calculate tumor volume, the modified ellipsoid formula, 1/2(length × width²), was used. Results from six injection sites were averaged. (b) Limiting dilution analysis of tumor development. Different numbers of ST1 or ST1‐N6 cells were injected, and the number of tumors at 4 and 8 weeks post‐injection per total number of injection sites was determined. Mice were sacrificed once a tumor exceeded 20 mm in diameter, which occurred before 8 weeks post‐injection in the ST1‐N6‐treated animals (indicated by –).

Figure 2

Upregulation of protein kinase B (AKT) signaling in ST1‐N6 cells. (a) Increased phosphorylation of AKT after serial transplantation. Cell lysates were prepared from each cell population and analyzed by Western blotting. Detection of α‐tubulin was used as a protein loading control. Band intensities were normalized to the intensity of α‐tubulin and then divided by the normalized intensity of the band in the parental ST1 cells. Representative results of three independent experiments are shown. (b) Western blot analysis of FOXO phosphorylation levels. Representative results of three independent experiments are shown. (c) mRNA level of the FOXO‐target gene TRAIL in ST1 and ST1‐N6 cells. The mRNA level was measured by real‐time RT‐PCR. Results from three independent samples were averaged and are presented with SD. **P < 0.01.

Figure 3

Correlation between the tumorigenicity and protein kinase B (AKT) activation of ST1 cells. (a) Western blot showing the exogenously expressed constitutively active form of AKT (CA‐AKT). Cells were first transduced with pRetroX‐Tet‐On Advanced retrovirus vector and then with control pRetroX‐Tight‐Pur (pTight) or a CA‐AKT coding retrovirus vector. The cells were cultured with (+) or without (−) doxycycline (Dox) and analyzed by Western blot. The immunoblot was probed with an antibody to S473‐phosphorylated AKT (pAKT) or total AKT. Exogenously expressed CA‐AKT (Exo) and endogenous AKT (Endo) are indicated. (b) Western blot analysis of the developed tumors. Control ST1 cells (No) or the retrovirus‐transduced cells described in (a) were injected into NOG mice, and the developed tumors were analyzed by Western blot. The blot probed with an anti‐hemagglutinin antibody (HA) showed the expression of CA‐AKT. (c) Tumor growth of control (pTight) or CA‐AKT vector‐transduced ST1 cells. Results from six injection sites were averaged. *P < 0.05. (d) Tumor growth by ST1‐N6 cells in NOG mice treated with the AKT inhibitor MK‐2206 or control vehicle (30% Captisol). Data shown are representative of two independent experiments. *P < 0.05.

Figure 4

Downregulation of inositol polyphosphate‐5‐phosphatase D (INPP5D) in ST1‐N6 cells. The expression level of INPP5D was assayed by real time RT‐PCR (a) and Western blotting (b). Results from three independent samples were averaged for the RT‐PCR analysis. **P < 0.01. (c) Increased protein kinase B (AKT) phosphorylation in ST1 cells after siRNA‐mediated gene knockdown of INPP5D. For the target gene, two siRNAs with different sequences were mixed (5 pmol, each siRNA) and introduced into ST1 cells (3 × 10⁶ in 100 μL) by electroporation. Cell lysates were prepared 48 h after electroporation and analyzed by Western blotting. No, cells without siRNA introduction; siNT, siRNA negative control.