p27kip1 expression limits H-Ras-driven transformation and tumorigenesis by both canonical and non-canonical mechanisms

The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation. Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin. Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner. Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice. Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.

A FACS analysis of typical cell cycle distribution observed in WT and p27KO control cells (vector) or H-Ras V12 -transformed fibroblasts, serum starved (T0) and then released in complete medium for the indicated times. B Western Blot analysis of Cyclin A (Cyc A), Cyclin B1 (Cyc B1), stathmin and p27 in WT and p27KO control (Vector) or H-Ras V12 -transformed fibroblasts, serum starved (0) and then released in complete medium (FBS) for the indicated times. Vinculin was used as loading control. C Graph reports the duplication time of WT and p27KO H-Ras V12 -transformed fibroblasts as evaluated in exponentially growing condition over a period of 4 days. Data report the mean of 2 ± SD of two different experiment performed with two different clones/genotype. Significance was calucluated using the Mann-Whitney unpaired test. D Matrigel evasion assay. Cells were included in a 3D-matrigel drop and incubated in complete medium for 48 hours. A typical image in phase contrast microscopy, using a 10X objective, is shown. Cells still included in the Matrigel drop (In) and cells already exited (Out) are indicated. E Transwell migration assay of WT and p27KO control cells (vector) or H-Ras V12transformed fibroblasts plated on transwell inserts coated with fibronectin and allowed to migrate up to six hours. Data represent the mean of 2 experiments performed in duplicate. F Wound healing migration assay of WT and p27KO control cells (vector) or corresponding H-Ras V12 -transformed fibroblasts, grown to confluence on plastic dishes and then scratched and allowed to migrate into the wound up to 24 hours. Typical images in phase contrast microscopy, using a 10X objective, are shown.
Supplementary Figure S3. Re-expression of human or mouse p27 proteins in p27 null H-Ras V12 fibroblasts have an equal impact on their phenotypes.
A Western Blot analysis of p27 and Ras expression in the indicated p27KO H-Ras V12 cell clones expressing mouse p27 WT or p27 CK-(mp27 WT or mp27 CK ) proteins. Vinculin was used as loading control. WT H-Ras V12 cells were used as control of endogenous p27 expression. B Western Blot analysis of p27 expression in the indicated p27KO H-Ras V12 clones expressing mouse (mp27 WT or mp27 CK ) or human p27 (hp27 WT ) proteins. Vinculin was used as loading control. C Growth curves of p27KO H-Ras V12 and p27KO H-Ras V12 transformed fibroblasts, re-expressing hp27 WT , mp27 WT or mp27 CK proteins, as indicated. Data display the number of cells at 5 days from plating and represent the mean of three different experiments +/-SD. D Soft agar assay of the indicated H-Ras V12 transformed fibroblasts. Data represent the mean (+/-SD) of three different experiments performed in duplicate in which 8-10 fields/well were scored. E Transwell migration assay of the indicated H-Ras V12 transformed fibroblasts plated on transwell inserts coated with fibronectin and allowed to migrate up to 8 hours. Data represent the mean of two experiments performed in duplicate. Figure S4. Low cytoplasmic p27/stathmin ratio is associated with hyper-activation of ERK1/2 pathway in human carcinomas and carcinoma-derived cell lines.

Supplementary
A Western Blot analysis of ERK1/2 phosphorylation status, and p27 and stathmin expression levels in two human colon carcinoma cell lines (non B-Raf mutated), serum starved and then stimulated with complete medium (FBS) for the indicated times. At each time point, cells were lysed to separate their nuclear (N) and cytoplasmic (C) fractions. B qRT-PCR analysis of Egr-1 expression in Colo-201 and Colo-205 cells serum starved and then stimulated with complete medium (FBS) for the indicated times. Data were normalized using the expression of an housekeeping gene and expressed as fold increase over the level of time zero. A.U., arbitrary units. Error bars represent SEM C Western Blot analysis of ERK1/2 phosphorylation status, and p27 and stathmin expression levels in two HER2 positive human breast carcinoma cell lines, serum starved and then stimulated with complete medium (FBS) for the indicated times. At each time point, cells were lysed to separate their nuclear (N) and cytoplasmic (C) fractions. In the right graph, the normalized ERK phosphorylation expressed as fold increase over the T0 (i.e. serum deprived cells) is reported. D Western Blot analysis of ERK1/2 phosphorylation (right panel) in SK-BR-3 mammary carcinoma cell line, following silencing of p27 with specific shRNAs (left panel). Control shRNAs were used to exclude transduction-related nonspecific effects. On the right, graph reports the quantification of ERK activation, normalized by total ERK expression in each corresponding lysate. E Western Blot analysis of ERK1/2 phosphorylation, p27 and stathmin expression in the cytoplasmic fractions of 17 representative human mammary carcinoma samples (out of 37 analyzed). Vinculin was used as loading control. The right graph reports the quantification of the western blot analysis of ERK1/2 phosphorylation in cytoplasm of 37 human breast cancer specimens, as listed in Supplementary Table 1. Tumors were segregated in two groups, displaying either low or high p27/stathmin ratio, then a value of normalized phospho-ERK1/2 expression was assigned to each sample.

Cell cultures
Primary wild type (WT), p27 knock-out (p27KO) and p27/stathmin double KO (DKO) mouse embryo fibroblasts (MEF) were prepared from embryos at day 13.5, according to standard procedures. The correct genotype of WT, p27KO and p27/stathmin DKO cells was determined by PCR, as described [6,7]. 3T3 fibroblasts were generated from primary MEFs, following the 3T3 immortalization protocol, as Colo-201 and Colo-205 human colorectal adenocarcinoma cells were cultured in RPMI-1640 supplemented with 10% FBS (Sigma).  Fresh medium with 1.5 μg/ml Puromycin and/or 0.4 mg/ml Hygromycin was added to the wells every 3 days. Where indicated, FTI inhibitor was added in the top agar and in the medium at the concentration of 6 μM. On day 15, the number of colonies was counted in 10 random fields at 10X magnification.

Motility assays
For 3D-Matrigel™ evasion assay, cells (7,5x10 5 /ml) were included in Matrigel™ (6 mg/ml, Becton Dickinson) drops and maintained 1 hour upside-down at 37°C. Then, complete medium was added and cell motility monitored by transmission microscopy, using a Nikon TS100/F microscope and images collected using a CCD camera (Leica). The evasion ability was estimated by measuring the distance covered by the cells from the drop edges, 5 days after inclusion. To perform this analysis, cells were stained with crystal violet and pictures were taken. After conversion from pixel to millimeters, the actual distance covered was calculated. Proteins were extracted from frozen tissues, as described in Supplementary Materials.

Isolation of RNA from cells, lung and circulating cells samples was performed using
RNeasy-Mini Kit (QIAGEN), according to the manufacturer's instructions.
Disruption of the tissue sample was achieved by grinding the frozen tissue thoroughly with lead blocks. The homogenization was performed passing the lysate at least 5 times through a blunt 23-gauge needle fitted to an RNase-free syringe. This approach was used to identify circulating tumor cells and/or metastatic cells. We want to highlight that although we extracted the RNAs from tissues and from circulating cells pellets and we have probably lost any circulating free nucleic acid in our preparations we cannot completely exclude that our approach could have also led to the amplification of some residual circulating RNAs.
RNA were then quantified and retro-transcribed with AMV Reverse transcriptase, according to manufacturer's instructions (Promega) and the obtained cDNAs were amplified with nested PCR, in order to evaluate the presence of ectopic cells in tissue samples.
The following primers were used: Standard curves (10-fold dilution from 10 1 to 10 -4 attomoles) were prepared both for target genes and for housekeeping genes. The incorporation of the SYBR Green dye into the PCR products was monitored in real time using the Applied Biosystems ABI PRISM 7700 Sequence Detector, and the resulting threshold cycles (Ct) were computed. Ct values were converted into attomoles and the normalized target gene value was obtained by using at least two different housekeeping genes.

Preparation of protein lysates, immunoprecipitation and immunoblotting
Proteins were collected in indicated culture conditions, such as exponential growth, high confluence, starvation in serum free medium and after release in complete medium (10% FBS) or adhesion to Fibronectin (10 g/ml, Sigma). Where indicated, cells have been also treated with the following compounds for the indicated time: FTI-276 (6 M, Sigma), Y27632 (10 M, Calbiochem), EGF (3 ng/ml, Invitrogen)..
To extract total proteins from cells, at the indicated time points, cells were scraped on ice using cold NP40 lysis buffer (0.5% NP40; 50 mM HEPES pH 7; 250 mM NaCl; 5 mM EDTA; 0.5 mM EGTA, pH 8), supplemented with a protease inhibitor cocktail (Complete™, Roche) and 1 mM Na 3 VO 4 (Sigma), 10 mM NaF (Sigma) and 1 mM DTT (Sigma). To extract total proteins from mouse organs or from tumor specimens, the same procedure was used, except that tissue disruption was first achieved by using the TissueLyser II (QIAGEN).
To perform the differential extraction of cytoplasmic and nuclear proteins, cells were resuspended in buffer A (10 mM HEPES pH 7.9, 0. Primary antibodies were purchased from BD: p27 (610242)

Ras Pull-Down
3T3 fibroblasts transformed with H-Ras V12 or K-Ras V12 ,or HT1080 cells transduced with expressing Ad-green adenoviruses encoding or not (control) for p27WT or p27CK-proteins, were lysed as described above and 2 mg of protein were incubated with glutathione S-transferase (GST)-Raf bound to glutathione-Sepharose high performance (GE Healthcare) for 1 h at 4°C. After thorough washes, the samples were boiled for 10 minutes in Laemmli buffer to detach active GTP-bound Ras, loaded on 15% SDS-PAGE gels (Criterion; Bio-Rad) and then immunoblotted using anti-FLAG antibody.

Immunofluorescence Analysis
For immunofluorescence analyses tumor specimens tissues embedded in OCT were sectioned with a cryostat and fixed with 4% PFA for 10 minutes at RT. After washing in PBS, sections were immersed into 10 mM citrate buffer pH 6.0 and antigens retrieved by boiling in the microwave (550 W, 20 minutes). Samples were slowly cooled down to RT and specimens permeabilized with 0.4% Triton X-100 (Sigma-Aldrich) in PBS for 10 minutes. Samples were then blocked with 10% normal goat serum in PBS with 0.1% Triton X-100 for 20 minutes, incubated ON at RT with anti-Ki67 polyclonal antibody (Abcam, Cambridge, UK) followed by incubation with anti-