SIK1 couples LKB1 to p53-dependent anoikis and suppresses metastasis

Abstract

Resistance to anoikis, the subtype of apoptosis triggered by lack of adhesion, contributes to malignant transformation and the development of metastasis. Although several lines of evidence suggest that p53 plays a critical role in anoikis, the pathway(s) that connect cell detachment to p53 remain undefined. Here, through the use of a kinome-wide loss-of-function screen, we identify the serine-threonine kinase SIK1 (salt-inducible kinase 1) as a regulator of p53-dependent anoikis. Inactivation of SIK1 compromised p53 function in anoikis and allowed cells to grow in an anchorage-independent manner. In vivo, SIK1 loss facilitated metastatic spread and survival of disseminated cells as micrometastases in lungs. The presence of functional SIK1 was required for the activity of the kinase LKB1 in promoting p53-dependent anoikis and suppressing anchorage-independent growth, Matrigel invasion, and metastatic potential. In human cancers, decreased expression of the gene encoding SIK1 closely correlated with development of distal metastases in breast cancers from three independent cohorts. Together, these findings indicate that SIK1 links LKB1 to p53-dependent anoikis and suppresses metastasis.

Fig. 1. Identification of SIK1 as a suppressor of AI-growth in HMECs

(A) Experimental outline for the kinome-wide shRNA screen that identified SIK1 as a suppressor of AI-growth of tHMEC-P cells. (B) AI colony formation of tHMEC-P cells expressing various cDNAs or shRNAs as indicated, and cells expressing shSIK1#1 with add-back of a wobble SIK1 (wb-SIK1). The number of colonies is per 60 mm plate. Results are shown as the mean ± SD for 3 independent experiments.

Fig. 2. SIK1 is required for p53-dependent anoikis

(A) p53 protein abundance in HMECs expressing shSIK1s or shLuc cultured under attached or single cell suspension conditions for 4 or 8 hours, were examined by immunoblot analysis. Vinculin used as a loading control. Results are shown as the mean ± SD for 3 independent experiments. * p <0.01 (t-test) (B) Immunoblot analysis of p53 abundance in various tHMEC-P cell lines as indicated, as well as in colony-derived tHMEC-P-shSIK1 cells treated with adriamycin (ADR) (4.5 hr). wb-SIK1 (wobble SIK1), or vector, was expressed in AI colony-derived tHMEC-P-shSIK1#1 cells as indicated. (C) Kinase assays were used to detect auto-phosphorylation of SIK1 and phosphorylation of GST-p53 or GST-p53 ^S15A by Flag-tagged SIK1-WT or SIK1-KD immunoprecipitates. Radioactive signals were detected by phosphorimaging. (D) Phosphorylation of p53 at Ser15 was determined by immunoblot following kinase assay using Flag-tagged SIK1 immunocomplexes (WT or KD) from U2OS cells subjected to different conditions. (E) PUMA and BAX expression in tHMEC-P cells expressing shSIK1#1, SIK1-KD, shTP53, or p53DD cultured attached or in suspension for 16 hours was determined by real-time RT-PCR. Mean ± SD for 3 independent experiments are shown. * p <0.01 (t-test) (F) Inactivation of SIK1 renders cells resistant to anoikis. tHMEC-P cells were cultured in suspension for 2 days, and the percentage of apoptotic cells expressing various cDNAs or shRNAs was determined by Annexin-V staining. Mean ± SD for 3 independent experiments are shown. * p <0.001 (t-test).

Fig. 3. Loss of SIK1 contributes to metastasis

(A) Orthotopic xenograft assay. 2 × 10⁶ cells from the indicated population were resuspended in a final volume of 120 μl and injected into each inguinal mammary fat pad of athymic mice. When the primary tumor size reached 1.8cm, mice were sacrificed (Experiment A) or subjected to surgical removal of the tumors (Experiment B). After 6 months, all of experimental mice were sacrificed and subjected to histopathological examination of the lungs. PT, primary tumor; LM, lung metastasis; ND, not determined. * micrometastases; ** macrometasteses. (B) Representative images for lung micrometastases by H&E staining (left panels), genomic FISH with mouse Cot1 DNA (red) and human Cot1 DNA (green) (middle panels), and IHC with anti-human cytokeratin (right panels). Upper and lower panels show pulmonary micrometastases from mice injected with tHMEC-P cells expressing SIK1-KD or shSIK1 as indicated. Scale bars, 50 mm. (C) Experimental metastasis assay. Representative fluorescence images of lungs of mice injected through tail veins with GFP-labeled tHMEC-P cells expressing various constructs. Number of foci in each lung was scored under fluorescence microscope and data are shown as mean ± S.E.M. (n=6-8), * p <0.001 (t-test). Scale bars, 0.5 mm. (D) Western analysis of p53 abundance in tHMEC-PR cells expressing shTP53, shSIK1#1, or SIK1-KD maintained in attached culture or derived from lung metastases in NOD/SCID mice.

Fig. 4. SIK1 activation suppresses AI growth of LKB1-deficient HMECs

(A) Colony formation assays of tHMEC-P cells expressing shLKB1 or shLuc. SIK1-CA, but not SIK1-KD, suppressed AI growth of AI-derived tHMEC-P-shLKB1 cells. Mean ± SD for 3 independent experiments are shown. (B) Western blot analysis of p53 and LKB1 abundance in tHMEC-P cells expressing shLKB1 grown in attached culture or AI-derived tHMEC-P-shLKB1 cells expressing SIK1-KD or -CA as indicated. Flag-tagged SIK1 (KD or CA) HMECs was probed with anti-Flag antibody.

Fig. 5. SIK1 links LKB1 to p53-dependent suppression of metastasis

(A) A549 cells expressing various versions of SIK1 with or without co-expression of shTP53 were subjected to Matrigel invasion assay. Mean ± SD for 3 independent experiments. (B) GFP-labeled A549 cells expressing various constructs as indicated were injected into tail veins of NOD/SCID mice and lung metastasis was assessed 6 weeks post injection. Representative fluorescence images of mouse lungs are shown. Lung metastasis burden was shown as indices pooled with each group of mice expressed as % (± SEM) over controls (n=6-9), * p < 0.001 (t-test). (C) Summary of LKB1-SIK1 pathway that mediates p53-dependent anoikis and suppresses metastasis.

Fig. 6. SIK1 correlation with clinical outcome in human cancer

(A) SIK1 expression in normal breast or tumor samples from the DF/HCC or the UCSF cohorts is shown in boxplots. The mean for each group is indicated by the black center line, the first and third quantiles (the inter-quantile range, IQR) are indicated by the edges of the grey area. The extreme values (within 1.5 times the IQR from the upper or lower quantile) are indicated by the ends of the lines extending from the IQR. The difference in log2 expression ratios between normal and tumor samples was tested with Welch's t-test, p < 0.001. (B) and (C) Kaplan-Meier plots were used to assess the association of the levels of SIK1 transcripts with development of distal metastases. Samples were grouped as described in Materials and Methods. a, the DF/HCC cohort of breast cancer patients (n=133). b, the UCSF cohort of breast cancer patients (n=112). c, the Rotterdam cohort of breast cancer patients (n= 286). d, the subset of DF/HCC breast cancer patients whose tumors are negative for p53 IHC staining (n=76). e, the subset of UCSF breast cancer patients whose tumors are negative for p53 IHC staining (n=57).