Dysregulating IRES-dependent translation contributes to overexpression of oncogenic Aurora A Kinase

Abstract

Overexpression of the oncoprotein Aurora A kinase occurs in multiple types of cancer, often early during cell transformation. To identify the mechanism(s) contributing to enhanced Aurora A protein expression, a comparison between normal human lung fibroblast and breast epithelial cells to nontumorigenic breast (MCF10A and MCF12A) and tumorigenic breast (MCF-7) and cervical cell lines (HeLa S3) was performed. A subset of these immortalized lines (MCF10A, MCF12A, and HeLa S3) exhibited increased levels of Aurora A protein, independent of tumorigenicity. The increase in Aurora A protein in these immortalized cells was not due to increased transcription/RNA stability, protein half-life, or cap-dependent translation. Assays utilizing monocistronic and dicistronic RNA constructs revealed that the 5'-leader sequence of Aurora A contains an internal ribosomal entry site (IRES), which is regulated in a cell cycle-dependent manner, peaking in G2/M phase. Moreover, IRES activity was increased in the immortalized cell lines in which Aurora A protein expression was also enhanced. Additional studies indicated that the increased internal initiation is specific to the IRES of Aurora A and may be an early event during cancer progression. These results identify a novel mechanism contributing to Aurora A kinase overexpression.

Implications: The current study indicates that Aurora A kinase contributes to immortalization and tumorigenesis.

Figure 1

Enhanced translation contributes to over-expression of Aurora A protein in a subset of immortalized cell lines. (A) Endogenous Aurora A protein levels were analyzed via Western blotting and normalized to Gapdh. The Aurora A/Gapdh ratio from each cell line was compared to the ratio from WI-38 which was set to one. n=6 ± standard deviations (SD) (B) Total RNA was isolated and analyzed via qRT-PCR with Gapdh as a reference target. Results were normalized to the Aurora A mRNA level in WI-38 cells. n=3 ± SD (C) The ratio of the normalized Aurora A protein levels to normalized Aurora A mRNA levels are shown. (D) Quantitation from Western blotting of Aurora A protein expression levels in cells treated for 0 to 12.5 hours with cycloheximide. Expression level of Aurora A protein in the untreated cells (0 hr) was normalized to 100. n=3 ± SD (E) UV detection readout of sucrose gradient fractionation of MCF12A and MCF-7 cells (top). Quantitation of Aurora A mRNA levels associated with the different gradient fractions. Levels were measured using qRT-PCR after isolation of total RNA from each fraction (bottom). * = p<0.05, ** = p<0.005, *** = p<0.0001, student’s t test.

Figure 2

Inhibiting cap-dependent translation initiation differentially affects Aurora A protein expression. (A) Translation of a Photinus luciferase reporter mRNA containing the β-globin 5′ leader was transfected into the six cell lines. Luciferase activity was measured and the mRNA quantitated by qRT-PCR after 7 hrs. Shown is the ratio of luciferase activity to luciferase RNA. n=3 in triplicate ± SD (B) Western blots of lysates from HeLa cells transfected with nonsense siRNA or siRNA targeting eIF-4E for 48 hr. n=3, ± SD (C) Western blots of lysates from HeLa cells transfected with a control vector or a plasmid encoding for mutant hypophosphorylated 4E-BP1 for 48 hr. n=3 ± SD (D) Western blots of lysates from WI-38, HMEC, MCF10A, MCF12A and MCF-7 cells transfected with nonsense siRNA, siRNA 1 targeting eIF-4E or siRNA 2 targeting eIF-4E for 48 hr. (E) Quantification of eIF-4E knockdowns in WI-38, HMEC, MCF10A, MCF12A and MCF-7 cells. n=3 ± SD, ** = p<0.005, *** = p<0.0001, student’s t test.

Figure 3

The 5′ leader of the Aurora A mRNA contains an IRES. (A) Dicistronic luciferase mRNA (left) containing the β-globin, Aurora A, CrPV, and EMCV 5′ leaders or IRES elements were transfected individually into HeLa cells. Luciferase activity is shown as the ratio of Photinus luciferase to Renilla luciferase (P:R) and is normalized to that obtained from the dicistronic mRNA containing the β-globin 5′ leader. n=3 in triplicate ± SD Monocistronic Photinus luciferase mRNA (right) containing the β-globin or Aurora A 5′ leader were translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. The initial level of Photinus luciferase activity from each monocistronic mRNA was normalized to 100. n=3 ± SD (B) Cell cycle progression following the release of a double thymidine block in HeLa cells was confirmed by flow cytometry analysis (left). HeLa cells transfected with dicistronic luciferase mRNA containing the β-globin and Aurora A 5′ leaders were synchronized in G1/S with a double thymidine block and released. Luciferase activity is shown as the ratio of Photinus luciferase to Renilla luciferase (P:R) and is normalized to that obtained from the dicistronic mRNA containing the β-globin 5′ leader at the 0 time point (right). n=3 in triplicate ± SD, *** = p<0.0001, student’s t test.

Figure 4

Aurora A IRES activity is increased in the subset of immortalized cell lines that exhibit enhanced Aurora A protein expression. A) Dicistronic mRNA containing the β-globin and Aurora 5′ leaders were transfected into each cell line. The P:R ratio obtained from the β-globin construct from each cell line was set to one. The P:R ratio from the Aurora A construct was normalized to β-globin. n=3 in triplicate ± SD (B) Schematic representation of cotransfections of an A or m7G capped monocistronic Photinus luciferase mRNA containing the Aurora A or β-globin 5′ leaders with an m7G capped Renilla luciferase mRNA. C) Normalized luciferase activity obtained from the A capped Photinus luciferase constructs are represented as a percentage of the normalized luciferase activity obtained from the m7G capped Photinus luciferase construct for the β-globin 5′ leader (left) or the Aurora A 5′ leader (right). n=3 in triplicate ± SD, * = p<0.05, *** = p<0.0001, student’s t test.

Figure 5

Additional breast epithelial cell lines demonstrate a correlation between Aurora A IRES activity and Aurora A protein expression. (A) Endogenous Aurora A protein levels were analyzed via Western blotting and normalized to Gapdh. The Aurora A/Gapdh protein ratio from each cell line was compared to the ratio from WI-38 which was set to one. n=3 ± SD (B) Total RNA was isolated from the four cell lines and analyzed via qRT-PCR with Gapdh as a reference target. Results were normalized to the Aurora A mRNA level in WI-38 cells. n=3 ± SD (C) The ratio of the normalized Aurora A protein levels to normalized Aurora A mRNA levels are shown. (D) Quantitation from Western blotting of Aurora A protein expression levels from the four cell lines treated for 0 to 12.5 hours with cycloheximide. Expression level of Aurora A protein in the untreated cells (0 hr) was normalized to 100. n=3 ± SD (E) Normalized luciferase activity from the A capped Photinus luciferase constructs are represented as a percentage of the normalized luciferase activity obtained from the m7G capped Photinus luciferase construct for the β-globin 5′ leader (top) or the Aurora A 5′leader (bottom). n=3 in triplicate ± SD, ** = p<0.005, *** = p<0.0001, student’s t test.