Targeting thioredoxin reductase 1 reduction in cancer cells inhibits self-sufficient growth and DNA replication

Abstract

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1's role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase alpha, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

Figure 1. Thioredoxin reductase (TR1) expression in mouse and human malignant cells.

Indicated cell lines were metabolically labeled with ⁷⁵Se, the resulting protein cell extracts electrophoresed and the gels exposed to a PhosphorImager (see Methods). Selenoproteins identified previously in cell extracts , are indicated by an arrow and name on the right of each panel. TR1 was also identified by western blotting in protein extracts of each cell line as shown in the lower panels. (A, left panel) Control (parental; NIH3T3) and DT cells. (A, right panel) NIH3T3 (control, parental cells used in generating DT cells), LLC1 (mouse Lewis lung cell carcinoma), ACHN (human kidney renal cell carcinoma), A549 (human lung non-small cell carcinoma), HCT116 (human colon cell adenocarcinoma) and SNB19 (human cell glioblastoma). NIH3T3 was used as an indicator cell line for comparison of TR1 levels in a normal cell line to the malignant cell lines. (B) DT/pU6-m3 and DT/siTR1. DT (obtained by overexpression of mutant k-ras in NIH3T3) cells were stably transfected with the pU6-m3 (control) vector or siTR1 knockdown vector, respectively (see Methods).

Figure 2. Morphology, growth in soft agar and growth rates of TR1-expressing and -deficient cells.

(A) Control (NIH3T3, parental), DT, DT/pU6-m3 and DT/siTR1 cells. Cells were grown on culture plates and photographed during exponential growth (see Methods). (B) Anchorage-independent growth of control, DT, DT/pU6-m3 and DT/siTR1 knockdown cells. One thousand cells were suspended in soft agar and grown for two weeks. Plates were then stained with INT overnight and photographed. Details are given in Methods. (C) Growth rates of control, DT/pU6-m3 and DT/siTR1 cells under normal and serum-deficient conditions. Control, DT/pU6-m3 and DT/siTR1 cells were seeded (2×10⁵ cells/60mm dish) and grown under normal growth conditions (left panel), and DT/pU6-m3 and DT/siTR1 cells were seeded (5×10⁵ cells/60 mm dish) and grown in serum-deficient medium (right panel). Growth rates in serum-deficient medium were compared to those obtained under normal growth conditions.

Figure 3. Cell cycle analysis.

Control, DT/pU6-m3 and DT/siTR1 cells were grown in serum-deficient medium for 0, 24 and 48 hrs and incubated with BrdU for 6 hours. Harvested cells were stained with anti-BrdU antibody to monitor newly replicated genomic DNA and 7-AAD to monitor whole genomic DNA. (A) Stained cells were analyzed by flow cytometry and (B) quantitated in each phase of the growth cycle by FlowJO. Phases of the growth cycle, G0-G1, S phase (Early S and Late S) and G2-M were shown and the values given represent the percent of the total cell population. Details of the experiments shown in the figure are given in Methods.

Figure 4. Analysis of components involved in DNA replication.

Expression levels of DNA polymerase components, DNA Pol α, β, δ and ε, Cdc45 and PCNA, in control, DT/pU6-m3 and DT/siTR1 cells were analyzed by western blotting. Cells were grown in serum-deficient medium for 0, 24 and 48 hrs, harvested, protein cell extracts prepared and electrophoresed as described in Methods.