LMO4 mRNA and protein are increased in ErbB2-induced tumors. (a) Tumor (n=5) and hyperplastic (n=4) mammary glands from MMTV-c-Neu mice were compared to wild-type mammary glands (3 pools of n=5) using gene expression microarrays. LMO4 mRNA was elevated 4.5 fold in tumors and 2.3 fold in hyperplastic glands when compared to wild-type glands. * p<0.05, ** p<0.005, respectively. (b) An antisense LMO4 probe was used for in situ hybridization of an MMTV-c-Neu tumor. Asterisk (*) indicates tumor tissue. Sense control probe hybridized to the same tumor is shown to the right. (c) Whole tissue lysates of mammary glands from age-matched wild type mice (n=3) and tumors (n=3) from MMTV-c-Neu mice were analyzed by western blotting. β-actin was used as loading control.

ErbB2 and PI3K activity are necessary for LMO4 expression. (a) Western blot analysis of LMO4 expression in BT-474 cells treated with the following small molecule inhibitors: ZD1836 (EGFR, 1 µM), AG825 (ErbB2, 20 µM), LY294002 (PI3K, 40 µM), PD98059 (MEK, 20 µM), and JNK Inhibitor II (JNK, 25 µM). β-actin was used as a loading control. (b) Quantification of (a). LMO4 was expressed relative to β-actin. Values are the average of at least 4 independent experiments. Error bars depict standard deviations. * p<0.05 and ** p<5×10⁻⁴ compared to vehicle. (c) Total RNA was isolated 12 hrs after treatment of BT-474 cells with AG825 (ErbB2, 20 µM) and analyzed for LMO4 mRNA expression using real time RT-PCR. Values represent the averages of at least 4 experiments. * p<5.0 × 10⁻⁷ compared to vehicle-treated cells. (d) (left panel) BT-474 cells were treated with vehicle or AKT Inhibitor V (AKT, 5 µM) for 24 hrs and then cell lysates were collected for immunoblotting against LMO4. phospho-AKT (pAKT) was used to demonstrate inhibitor efficacy while total AKT and β-actin were used as loading controls. (right panel) HEK-293T cells were transiently transfected with 1.5 µg of myristolated AKT (myr-AKT), kinase inactive AKT (K179M AKT) or empty vector. Cells were lysed after 48 hrs and analyzed for LMO4 protein expression. p-AKT and total AKT levels are shown to confirm plasmid activity. β-actin was used as loading control. (e) (upper panel) Schematic of the LMO4 gene which has 5 exons (E1-E5) and two promoters (P1 and P2). The alternative transcription start sites yield identical protein products because the translation start site (ATG) is in Exon 2. The 5’ and 3’ ends of each construct are indicated below each promoter. (bottom panel) Activity from two proximal LMO4 promoter constructs (P1) and one distal LMO4 promoter construct (P2) was assessed in BT-474 cells treated with various inhibitors. All luciferase values were first normalized to a renilla transfection efficiency control and then expressed relative to values obtained with a minimal SV40 promoter. * p<5×10⁻⁴, ˆ p<0.05, ˆˆ p<0.001.

LMO4 regulates proliferation of ErbB2-dependent breast cancer cells. BT-474 and SKBR3 cells were transiently transfected with non-silencing siRNA (NS), or an siRNA directed to LMO4 (siLMO4#3). 36 hrs after transfection, cells were pulsed with BrdU for 1 hr. The percentage of BrdU positive cells was determined by immunofluorescence. (a,c) Immunoblotting was performed to confirm knock-down of LMO4 in BT-474 cells (a) and SKBR3 cells (c). β-actin was used as loading control. (b,d) Quantitation of proliferation rate was performed by calculating the percentage of BrdU-positive cells in a minimum of three independent experiments. Values are means ± standard deviations. * p<0.05, ** p<0.01, compared to non-silencing siRNA control.

LMO4 is required for maintenance of Cyclin D1 mRNA and protein expression in cells with constitutive ErbB2 signaling as well as for heregulin-induced upregulation of Cyclin D1. (a,b) Analysis of changes in Cyclin D1 and Cyclin E protein levels in response to LMO4 silencing. (a) BT-474 (left panel) and SKBR3 (right panel) cells were transiently transfected with non-silencing siRNA (NS) or siRNA directed to LMO4 (siLMO4#3) and whole cell lysates were collected 36 hrs post-transfection. Representative western blots for Cyclin D1 and Cyclin E are shown. β-actin was used as a loading control. (b) Quantitation of three independent experiments performed in BT-474 cells, each with at least 2 independent replicates. Values are means ± standard deviation. ˆ p<5×10⁻⁵, * p<0.001. (c,d) Analysis of changes in Cyclin D1 mRNA expression in response to changes in LMO4. RNA was isolated 24 hrs after transfection of BT-474 cells with non-silencing siRNA (NS), or two different siRNA directed to LMO4 (siLMO4#1 and siLMO4#3), and analyzed by real time RT-PCR. Values were expressed relative to GAPDH and normalized to non-silencing control. (c) Bars represent means ± standard deviations from 3 separate experiments each with 6 independent replicates. * p<0.05 (d) Scatter plot analysis of representative experiments from (c) including linear regressions. The correlation coefficient between Cyclin D1 mRNA and LMO4 mRNA is 0.93. (e,f) MCF-7 cells were incubated for 24 hrs with either 5 ng/mL of heregulin (hrg) or vehicle following transient transfection with an LMO4 siRNA (siLMO4#3) or a non-silencing siRNA (NS) control. (e) Representative western blot (f) Bars represent the means ± standard deviations of at least 3 independent experiments. The Cyclin D1 fold induction between NS and siLMO4#3 treated cells was significantly different (* p<0.05).

LMO4 regulates G2/M transition by inducing cullin-3 expression. (a,b) BT-474 (a) and SKBR3 (b) cells were transfected with a non-silencing (NS) siRNA or siRNA directed to LMO4 (siLMO4#3). Thirty-six hours after the end of transfection, cells were stained with propidium iodide and examined by FACS. The graphs depict the percent of cells in subG1, G1, S and G2/M stages of the cell cycle. * p<0.05, ** p<0.01, *** p<0.005, compared to non-silencing control. (c) BT-474 cells were harvested 12 hrs after RNAi transfection. Suppression of LMO4 protein was confirmed and mRNA samples were analyzed by an RT-PCR array of 84 cell cycle genes. The graph plots the average fold change (Log2)vs. p-values for all 84 genes from 3 independent experiments, each with two replicate samples. Genes located outside the vertical, dashed black lines are changed more than 2 fold. Values above the horizontal, dashed grey line are statistically significant at p <0.05, compared to non-silencing control. ▪ represents the cullin-3 mRNA (downregulated by 25 fold, p<0.02) (d) Semi-quantitative RT-PCR was used to confirm cullin-3 (cul3) mRNA downregulation after silencing LMO4 in three additional experiments. β-actin was used as an internal control. (upper panel) Representative gel. (lower panel) Average cul3 mRNA expression relative to β-actin ± standard deviation. * p<0.05. (e) BT-474 cells were treated with vehicle or AG825 (20 µM) for 12 hrs. RNA was isolated and analyzed by semi-quantitative RT-PCR for cullin-3 (cul3) and β-actin mRNA expression (upper panel) Representative gel. (lower panel) Bars are the mean cul3 mRNA levels relative to β-actin mRNA ± standard deviations in 4 independent experiments. ** p<0.005, compared to vehicle control.

LMO4 expression oscillates throughout the cell cycle with peak expression occurring at G2/M phase. HEK-293T cells were synchronized using aphidicolin (1.5 µg/mL). After re-initiation of the cell cycle with removal of aphidicolin and addition of serum, cells were harvested at the indicated times for western blotting and flow cytometry. (a) Representative western blot showing LMO4 protein levels at various time points after aphidicolin release. β-actin was used as loading control. (b) Quantitation of LMO4 protein expression (upper panel) and cul3 mRNA expression (lower panel) from a representative experiment. LMO4 values were normalized to β-actin. Cul3 mRNA levels were determined using real-time RT-PCR and were normalized to GAPDH. These experiments were repeated at least three times with similar results. * p<0.05 compared to baseline values, ** p<0.05 compared to values after completing one cell division. (c) Flow cytometry analysis of propidium iodide-stained cells for each of the indicated time points shows progression through one complete cell cycle. Areas representing cells at G1, S and G2 phases of the cell cycle have been annotated.

LMO4 is an essential intermediate of ErbB2-induced proliferation. LMO4 is target of ErbB2 signaling that induces proliferation. This pathway begins with ErbB2 regulation of PI3K activity, which controls expression of the transcriptional modulator LMO4 and ultimately leads to induction of cullin-3 gene expression. Cullin-3 in turn modulates cell cycle progression through G2/M. By regulating progression through G2/M, LMO4 also indirectly regulates expression of the G1 cyclins, Cyclin D1 and Cyclin E.