Increased Myc protein stability associated with increased pS62 and decreased pT58 in breast cancer cell lines. (A) [³⁵S]Methionine pulse/chase analysis shows the decay of radiolabeled Myc. Myc half-life was determined as described in SI Methods. (B) Western analysis of Myc decay in cells treated with 10 μg/mL cycloheximide (CHX). (C) Summary of Myc half-life in control cell lines and in breast cancer cell lines. Bars represent SD. P value was calculated by using a one-tailed Student t test. (D) Western analysis of pS62 and pT58 levels in the indicated cells. Total Myc antibody and pS62-Myc antibody were probed on one Western blot and detected by two-channel imaging with a LI-COR scanner. pT58 was probed separately. Expression of pS62/total Myc (left y axis) and pT58/total Myc (right y axis) was quantified and normalized to MCF10A. Bars represent SD (*P < 0.05, **P < 0.01, and ***P < 0.001).

Increased pS62 and decreased pT58 levels of Myc in human breast cancer. (A) Matched sections of tumor and normal tissue from patient 1 were placed on the same slide and simultaneously stained with pS62 antibody (red) and DAPI (blue). (Scale bars: 50 μM.) (B) Serial sections from patient 1 were stained with pT58 antibody (red) and DAPI (blue). (Scale bars: 50 μM.).

Decreased Axin1 in human breast cancer. (A) Quantitative PCR (qPCR) analysis of AXIN1 versus ACTIN expression tumor samples relative to matched normal samples was graphed in the order of most down-regulated AXIN1. In this case only, bars represent SD from the triplicate qPCR reactions. Missing sample numbers are a result of a lack of sufficient cDNA. The matched normal/tumor ratios were log-transformed, and a P value for AXIN1 down-regulation significance was calculated by single-tailed t test (P = 0.005). (B) qPCR analysis of AXIN1 mRNA levels in human breast cancer cell lines relative to MCF10A cells. (C) Western analysis of Axin1 protein levels in breast cancer cell lines and control MCF10A cells. (D) MDA231 cells were treated with 5 μM IWR-1 for 4 h and harvested for qRT-PCR analysis of Myc and Western analysis of the indicated proteins. MYC mRNA from equal total RNA input was graphed based on changes in Ct and is shown ±SD. (E) MDA231 cells were starved in 0.1% FBS for 48 h, treated with DMSO or 10 μM IWR-1 for 1 h, and stimulated with 10% FBS for another 3 h in the presence of DMSO or IWR-1. Protein stability was analyzed as described in Fig. 1 (*P < 0.05, **P < 0.01, and ***P < 0.001).

Axin1 regulates Myc oncogenic activity in breast cancer. (A) The MCF10A stable clones Ctrl#1,2 (empty vector) and shAxin1#4,6 were grown in soft agar for 4 wk with or without 1 μg/mL doxycycline (Dox), and colonies were counted. The degree of Axin1 knockdown in different clones is shown in the Western analysis. (B) Stable clones were infected with adenovirus Ad-Myc^T58A for 18 h. Soft agar assay was done as in A. (C) SKBR3 cells were treated with 10 μM IWR-1 for 24 h and analyzed for Myc and Axin1 expression by Western analysis. MYC mRNA expression was analyzed as in Fig. 3D. (D) SKBR3 cells were starved in 0.2% FBS for 24 h and treated with 5 μM IWR-1 or DMSO for another 30 h. Cells were treated with 10 μM MG132 for 4 h before harvesting for coimmunoprecipitation (co-IP) with Myc antibody C33. Western analysis of input and IP is shown. (E) Cell growth curve of SKBR3 cells with or without IWR-1 treatment. (F) Soft agar assay of SKBR3 cells treated with 10 μM IWR-1 or DMSO as described in Methods. (G) SKBR3 cells were treated with 10 μM IWR-1 for 4 h, and ChIP was done with N262 antibody followed by qPCR. GAPDH internal primers were used as negative control (Neg. ctrl). Western blot of ChIP inputs is shown at the top right corner (*P < 0.05, **P < 0.01, and ***P < 0.001).

Switch from AXIN1V1 to AXIN1V2 expression in breast cancer. (A) Schematic diagram of Axin1v1 and Axin1v2 coding exons modified from a previous publication (28). (B) qRT-PCR analysis of AXIN1V1 and AXIN1V2 in breast cancer cell lines. Ratios of AXIN1V1 versus total AXIN1 and AXIN1V2 versus total AXIN1 normalized to those of MCF10A were graphed ±SD. (C) Sets of normal (N) human breast tissue and matched tumor (T) tissue were prepared as snap-frozen samples for extracting RNA for qRT-PCR of AXIN1V1 and AXIN1V2 expression or paraffin-embedded samples for pS62 immunofluorescence. The ratio of V2 versus V1 and the pS62 staining intensity in tumor tissue relative to matched normal tissue were graphed. Correlation coefficient was calculated by using Excel. (D) Top: SNU475 cells were infected with lentivirus expressing V5-tagged LacZ as a control or V5-tagged Axin1v1 or v2 protein for 72 h and subjected to Western analysis. Graph shows pS62-Myc versus Actin levels from multiple experiments. (E) SNU475 cells were infected as in D for 48 h and then infected with adenovirus expressing HA-tagged Myc for 18 h. ChIP studies were done with HA antibody as described for Fig. 4G. (F) A model showing deregulation of Axin1 contributes to stabilization and accumulation of pS62-Myc in breast cancer. In normal cells, Axin1 coordinates a Myc destruction complex including GSK3β, PP2A, and other proteins, and promotes Myc degradation. In tumor cells, a switch in Axin1 splice variants and/or decreased Axin1 total level contributes to disruption of the Myc destruction complex and resulting accumulation of pS62-Myc. Fig. S8 shows a more detailed model (*P < 0.05, **P < 0.01, and ***P < 0.001).