Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer

Abstract

Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. As LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we show that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1 null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

Figure 1. Endogenous Crtc1 in normal and human tumor cells

a) N-terminal anti-Crtc1 antisera detects the recombinant Crtc1-Maml2 fusion peptide (C-M2) transfected into H2009 lung cancer cells and the endogenous C-M2 fusion peptide (H3118 lung mucoepidermoid cells) at 135 kDa (upper arrow). Steady-state expression of the endogenous 85 kDa Crtc1 is detected in both cell lines (lower arrow). b) Variable expression of endogenous Crtc1 in brain tumor samples. Glioblastoma (GBM), astrocytoma (Astro), oligodendroglioma (Oligo), grade II/III (II/III).

Figure 2. Endogenous and recombinant Crtc1 mobility pattern linked with LKB1 status in human tumor cells

a) 60 μg protein extracts subjected to imunoblotting using peptide anti-Crtc1 antisera. LKB1 protein status is indicated above each lane and was confirmed by imunoblotting for each sample (data not shown). The samples on the left panel were run on a gradient gel and on the right panel on a 7% acrylamide gel to emphasize the size differences. Arrows depict the faster and slower Crtc1 mobility forms. b) Flag-tagged wildtype (wt) Crtc1 and the indicated point mutant Crtc1 vectors were transfected into either LKB1 wildtype cells (H3118) or LKB1 null cells (H2126) and protein extracts were subjected to immunoblotting with anti-Flag. c) Flag-tagged wt Crtc1 was transfected in LKB1 wildtype cells and subjected to incubation with forskolin 10 microgram/1 hr (FSK10) or 60 microgram/4 hrs (FSK60) treatment as indicated. d) H3118 cells were repeated as in panel c and immunoblotted with anti-Crtc1 antisera. e) LKB1-null H2126 cells were mock-transfected or transfected with either the kinase dead (KD) or wt LKB1 plasmids and protein extracts subjected to imunoblotting with either anti-Crtc1 (top) or anti-LKB1 (bottom).

Figure 3. LKB1 null status associated with faster Crtc1 mobility

a) mesothelioma tumor cells lines were subjected to imunoblotting with anti-LKB1. b) High resolution CGH analysis for LKB1 detects homozygous deletion within LKLB1 locus exclusively for H2369 cells. c) matched tumor cells lines subjected to immunoblotting with anti-Crtc1.

Figure 4. Enhanced nuclear staining of endogenous Crtc1 in LKB1 null cells

a) H2087 and H522 LKB1 wildtype cells and b) H2126 and H23 LKB1 null cells were stained with DAPI and anti-Crtc1 in the absence or presence of the blocking immunogenic Crtc1 peptide and visualized by immunofluorescence.

Figure 5. Activation of the Crtc1/CREB target gene NR4A2/Nurr1 in LKB1 null samples

a) RT-PCR for the indicated target genes and GAPDH in cells carrying the Crtc1-Maml2 fusion (*C-M2) and LKB1 wildtype (+) and LKB1 null (−) tumor samples. b) Quantitative RT-PCR for NR4A2/Nurr1 in LKB1 wildtype (H2087) and LKB1 null (H2126 and H460) samples. The NR4A2 signal was normalized for GAPDH expression. c) Protein immunoblot analysis for NR4A2 in tumor samples with indicated matched LKB1 status.

Figure 6. NR4A2 RNAi colony growth inhibition

a) NR4A2 immunoblot showing RNAi protein knock-down by either control pSHAG RNAi #3 or pSHAG #1 sequences or by NR4A2 specific pSHAG RNAi vector. b) crystal violet staining of 10 cm dishes with indicated tumor lines grown 4 weeks in G418-containing media following transfection with mock (−), control pSHAG #3 RNAi (#3), or pSHAG NR4A2 RNAi plasmids. c) colony growth suppression data (mean and standard deviation) for pSHAG NR4A2 RNAi as compared with control RNAi transfection on the indicated tumor cell lines.