Figure 1.
RASSF1C stimulates lung cancer cell proliferation.
NCI-H1299 cells stably transduced with MLV-backbone (BB) or MLV-HA-RASSF1C (1C) were treated with 1 µg/ml doxycycline for 72 h, and then cells were counted. RASSF1C over-expression (1C) increased cell proliferation by 2.5 fold compared to control (BB). Data is representative of at least 3 independent experiments, and the values represent the mean±SEM. * P<0.05.
Figure 2.
. RASSF1C promotes lung cancer cell migration. (A) The BD BioCoat Matrigel Invasion Chamber was used to assess cell invasion /migration of NCI-H1299 cells stably transduced with empty MLV-backbone (BB) or MLV-HA-RASSF1C (1C). Cells treated with doxycycline at 1ug/ml were co-incubated with serum-containing media. After 24 h, the lower sides of the filters were fixed and stained, and cells in four microscopic fields were counted. The average cell number count was plotted. (B) NCI-H1299 cells over-expressing RASSF1C showed a higher number of cells invading the Matrigel chamber and migrating to the other side of the filter compared to control NCI-H1299-BB cells. Data is representative of at least 3 independent experiments, and the values represent the average cell colony count. * P<0.05 as determined by t-test.
Figure 3.
CD133+ cells expressing RASSF1C form tumor spheres more robustly than control cells.
(A) Non-SP-1C CD133- , SP-BB CD133+ , and SP-1C CD133+ cells isolated from the A549 lung cancer cell line were grown in serum free medium supplemented with 10 ng/ml EGF and 20 ng/ml FGF to promote tumor sphere formation for three wk. (B) A549 cells repopulated from tumor spheres were checked for HA-RASSF1C expression using HA-antibody. As expected, SP-1C CD133- and CD133+ expressed HA-RASSF1C, while SP-BB cells did not. Data is representative of at least 3 independent experiments.
Figure 4.
RASSF1C decreases the sensitivity of lung cancer cells to betulinic acid.
A549 (A) and NCI-H1299 (B) lung cancer cells over-expressing RASSF1C were treated with betulinic acid (BA) for 24 h. Cells were then assayed for cell viability/proliferation. RASSF1C over-expression significantly reduced the sensitivity of cells to BA. Data is representative of at least 3 independent experiments, and the values represent the mean±SEM. * P<0.05: for 1C compared to BB.
Figure 5.
Betulinic acid effects on Piwil1 gene expression.
Piwil1 mRNA expression was assessed in the presence and absence of serum and BA in the lung cancer cell lines A549 (A) and NCI-H1299 (B). RT-PCR data show that piwil1 expression was down-regulated by BA in the absence and presence of serum. PCR reactions were set in triplicates and Cyclophillin was used as an internal loading control and used to normalize the relative expression levels using the 2−ΔΔ method (26). RT-PCR reaction were run at three independent times.
Figure 6.
PIWIL1 silencing reduces β-catenin levels in lung cancer cells.
Knock-down of piwil1 expression resulted in a reduction in β-catenin mRNA and protein levels. (A) Western blot analysis of H1299 lung cancer cells infected with Mission Lentiviral-shRNA-control particles (shRNA-cont) and Mission Lentiviral-shRNA-piwil1 (shRNA-piwil1) to silence endogenous piwil1 expression. Western blot analysis using anti-PIWIL1 antibody shows reduced PIWIL1 protein levels in shRNA-PIWIL1 cells compared to shRNA-cont cells. Silencing of piwil1 gene expression resulted in a reduction in β-catenin protein levels as determined using β-catenin antibody. Normalized PIWIL1 protein levels to actin (loading control) are also shown, data represents three independent blots. (B) Piwil1 and β-catenin mRNA levels were also assessed in shRNA-cont and shRNA-piwil1 cells and the RT-PCR data show down-regulation of both piwil1 and β-catenin mRNA levels which is consistent with the Western blot analysis. RT-PCR experiments were carried out at least 3 independent times.
Figure 7.
Effect of RASSF1C-IGFBP5 interaction on PIWIL1 mRNA expression.
Piwil1 mRNA expression was assessed in H1299 cells stably transduced with MLV-HA-back bone (BB), MLV-HA-RASSF1C (1C), MLV-HA-RASSF1A and 1C (1A-1C) and MLV-HA-1C and IGFBP-5 (1C-BP5). RT-PCR data show that piwil1 mRNA expression in cells co-expressing 1C and BP5 was reduced compared to cells over-expressing 1C and 1A and1C. Data is representative of at least 3 independent experiments, and the values represent the mean±SEM. * P<0.05: for 1C compared to BB.
Figure 8.
Effect of RASSF1C-IGFBP5 interaction on PIWIL1 protein levels.
(A) Western blot analysis of NCI-H1299 and A549 cells stably transduced with MLV-backbone (BB), MLV-HA-RASSF1C (1C), MLV-HA-IGFBP-5 (BP5), and co-transduced with both MLV-HA-RASSF1C and –IGFBP-5 (1C-BP5) and treated with 1 µg/ml doxycycline for 48 h. The anti-HA tag antibody detected a HA-RASSF1C and HA-IGFBP-5 fusion protein in cells that over-expressed each alone and those that co-expressed them both. The fusion protein was visualized using fluorescently labeled secondary antibodies. (B) Western blot analysis of A549 and H1299 lung cancer cells stably transduced with MLV-backbone (A549-BB and H1299-BB), MLV-HA-RASSF1C (A549-1C and H1299-1C), MLV-HA-IGFBP-5 (A549-BP5 and H1299-BP5), and MLV-HA-RASSF1C/MLV-HA-IGFBP-5 A549-1C-BP5 and H1299-1C-BP5). The anti-PIWIL1 antibody was used to probe the Western blot. PIWIL1 is up-regulated in A549 and NCI-H1299 cells over-expressing RASSF1C, but not in cells that express IGFBP-5 or co-express both IGFBP-5 and RASSF1C. (C) Shows normalized PIWIL1 protein levels to actin (loading control) with standard deviations, data represents three independent blots.
Figure 9.
Co-expression of RASSF1C-IGFBP5 sensitizes cells to betulinic acid
. H1299-BB, H1299-1C, H1299-1A-1C, and H1299-1C-BP5 cells were treated with betulinic acid (BA) for 24 h. Cells were then assayed for cell viability/proliferation. Cells co-expressing RASSF1C-IGFBP-5 were more sensitive to BA compared to cells over-expressing RASSF1C or co-expressing RASSF1A-RASSF1C. Data is representative of at least 3 independent experiments, and the values represent the mean±SEM. * P<0.05: for 1C compared to BB.
Figure 10.
Dorsomorphin and Trichostatin A have opposite effects on RASSF1C and PIWIL1 gene expression.
H1299 lung cancer cells were treated with Dorsomorphin and Trichostatin A at a concentration of 10 µM to validate the PCR array data. RT-PCR analysis shows that Dorsomorphin significantly up-regulated RASSF1C gene expression while slightly up-regulating PIWIL1 gene expression. Trichostatin A treatment significantly down-regulated RASSF1C and PIWIL1 mRNA levels. The data presented is an average of three independent RT-PCR experiments done in triplicate, * = P<0.05.
Table 1.
PCR array screen.
Figure 11.
Proposed RASSF1C mechanistic pathway for lung cancer growth and progression.
This model is based on our (12) and others previous published work and current data presented in this article. RASSF1C activates the MEK-ERK1/2 pathway (12), which controls a wide variety of genes that promote cell division and proliferation. Over-expression of RASSF1C up-regulates Piwil1 gene expression (12). Treatment of H1299 lung cancer cells with the MEK-ERK1/2 pathway inhibitor CI1040 resulted in down-regulation of Piwil1 mRNA levels (12). PIWIL1 is widely over-expressed in tumors compared to normal tissue, and it may have important functions in cancer initiation, maintenance, or progression (increased epigenetic gene modification, proliferation and cell migration, and reduced apoptosis). PIWI-like proteins have been shown to interact with PIWI-interacting RNAs (piRNAs) to form complexes that regulate transcriptional and translational repression leading to inhibition of apoptosis, stimulation of cell division and proliferation, and down-regulation of cyclin inhibitors and tumor suppressors (18). Over-expression of PIWIL1 down-regulates IGFBP-5 (18), which is an interacting partner and inhibitor of RASSF1C (19). Thus, co-expression of RASSF1C and IGFBP-5 abrogates the up-regulation of PIWIL1 expression in response to increased RASSF1C, suggesting that IGFBP-5 could block RASSF1C actions through the ERK1/2 pathway resulting in reduced PIWIL1 expression and function. Also, RASSF1C mRNA expression is regulated by AMP and ATM kinases. Various key components of the proposed RASSF1C pathway will be tested to validate this model.