MUC1-C nuclear localization drives invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway

Abstract

MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1-CT are involved in increase of migration, cell viability, resistance to anoikis and in decrease of cell aggregation in cancer cells. Invasiveness depends only on MUC1-CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization drives invasiveness of cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies.

Figure 1. MUC1 increases migratory and invasive properties and decreases cell-cell interaction in ACHN and 786-O cells

Western blotting were performed with anti–MUC1 targeting VNTR extracellular domain (M8) or cytoplasmic tail (Ab-5), and anti–β-actin antibodies on whole cell extracts obtained from (A) ACHN clones stably transfected with different expression vectors: MUC1-Full Length (MUC1FL), -deleted for its Tandem Repeat domain (MUC1ΔTR) or -deleted for its Cytoplasmic Tail (MUC1ΔCT) or an empty vector (EV) or (B) from 786-O clones stably transfected with a shRNA control (scramble) or with shRNA targeting MUC1 (sh1.1 and sh1.2). Cell migration of ACHN (C) and 786-O (D) clones was evaluated using 24-well migration chambers with 10% fetal calf serum as chemoattractant. The values obtained in EV-ACHN and scramble 786-O control cells were referred to as 100. The graphs show a percentage of control migration 24h after seeding. Values are means s.e.m (standard error mean) and represent five separate experiments (*** p<0.001). Cell invasion ((E) ACHN clones and (F) 786-O clones) was evaluated using 24-well Matrigel® invasion chambers with 10% fetal calf serum as chemoattractant. The values obtained in EV-ACHN and scramble 786-O control cells were referred to as 100. The graphs show a percentage of control invasion 24h after seeding. Values are means s.e.m and represent five separate experiments (*** p<0.001). ACHN (G) and 786-O (H) clones were seeded on agarose 0.8% under shaking. After 1h, aggregated and isolated cells were counted to determinate % of cell aggregation. Values are means s.e.m and represent at least three separate experiments (** p<0.01).

Figure 2. MUC1 increases cellular viability and confers anoikis resistance

ACHN (A, C) and 786-O (B, D) clones were seeded in 96-wells plate coated (C, D) or not (A, B) with PolyHema and incubated at 37°C. By a MTS assay, cellular viability was assessed everyday for viability and after 5 days for anoikis. OD was read at 490nm. For anoikis assay, mean in EV-ACHN and 786-O scramble clones was arbitrarily set to 100. Values are means s.e.m and represent five separate experiments (* p<0.05, ** p<0.01).

Figure 3. MUC1FL increases cell growth in vivo

Subcutaneous injections with ACHN clones stably transfected with an empty vector (EV; ), with MUC1FL- (), with MUC1ΔTR- () or with MUC1ΔCT ()-expression vectors were performed in SCID mice. Values are means s.e.m and represent values obtained in 6 mice (* p<0.05, ** p<0.01).

Figure 4. Impact of MUC1 expression on signaling pathways

(A) Western blotting on whole cell extracts obtained from EV- and MUC1FL-ACHN clones or from 786-O scramble, sh1.1 and sh1.2 clones were performed. Antibodies against Bax, Bcl-xL, caspase 9, c-Myc, Akt, phospho-Akt, p38, phospho-p38, ERK, phospho-ERK, JNK, phospho-JNK, cyclin D1, β-catenin and β-actin were used. (B) Expression of NF-κB p65 and p50 subunits in cytosolic and nuclear extracts was carried out by western blot. (C-D) Luciferase activity of the κB-Luc synthetic promoter was measured 48h after transfection. Luciferase activity in EV-ACHN and scramble 786-O cells was set as 1. Values are means s.e.m and represent five separate experiments (*** p<0.001).

Figure 5. Increase of invasive properties mediated by nuclear MUC1-C is dependent of ADAM10/ADAM17/γ-secretase activities

(A) Western blotting was performed on nuclear fraction of ACHN cells after treatment by different siRNA. The intensities of the signals were determined by densitometric scanning and are expressed as the relative signal intensity compared with that obtained with control siRNA. (B) Expression of MUC1-CT in cytosolic and nuclear fractions was carried out by western blotting from EV- and MUC1FL-ACHN clones treated or not with 10 ng/ml of L685,458, a γ-secretase inhibitor. (C) Cell invasion of ACHN cells transfected with different siRNA or treated 24h with 10 ng/ml of L685,458 was evaluated using 24-well Matrigel® invasion chambers with 10% fetal calf serum as chemoattractant. The graphs show the total number of invasive cells counted 24h after seeding. Values are means s.e.m and represent five separate experiments. (D) Invasion experiment was also performed on ACHN cells treated with 5 μM of CP-2 (control) or GO-203 peptides. Values are means s.e.m and represent five separate experiments (** p<0.01, *** p<0.001)

Figure 6. MUC1 is involved in increase of ADAM10 and ADAM17 expression

Western blot performed on cell extracts obtained from EV- and MUC1FL-ACHN clones treated or not by 5 μM of CP-2 and GO-203 peptides (A), scramble, sh1.1 and sh1.2 786-O clones (B) or primary proximal tubular epithelial cells (PTE), RCC4, RCC10 and 786-O cancer cell lines (C). The density of each marker was measured and ADAM10/actin and ADAM17/actin ratios were determined and represented as histograms. Expression in control cells was arbitrarily set to 1.

Figure 7. MUC1, ADAM10 and ADAM17 expression in human tissue

Immunohistochemical expression of MUC1, ADAM10 and ADAM17 assessed in human normal kidney (up) and a human cRCC (down). In normal kidney, MUC1 staining restricted to apical surface (insert) of distal convoluted tubules, membranous ADAM10 staining is (insert) on distal convoluted tubules and negative ADAM 17 staining. In cCRCC, diffuse cytoplamic MUC1 staining, nuclear ADAM10 staining and focal and weak ADAM17 staining (insert) of tumor cells (Magnification, × 200; x400 (insets)).