Vimentin regulates differentiation switch via modulation of keratin 14 levels and their expression together correlates with poor prognosis in oral cancer patients

<div><p>Vimentin is an intermediate filament protein, predominantly expressed in cells of mesenchymal origin, although its aberrant expression is seen in many carcinomas during epithelial mesenchymal transition. In cancer, vimentin expression is associated with the transition from a more differentiated epithelial phenotype to a dedifferentiated state. In view of the perceived role of keratins (Ks) as regulators of differentiation in epithelia, it was important to understand whether vimentin modulates differentiation through the reprogramming of keratins, in transformed cells. To address this, vimentin was stably downregulated in oral cancer derived cells. Further, global keratin profiling was performed after high salt keratin extraction. K5/K14 pair was found to be significantly downregulated, both at protein and mRNA levels upon vimentin downregulation. The previous study from our laboratory has shown a role of the K5/K14 pair in proliferation and differentiation of squamous epithelial cells. Vimentin depleted cells showed an increase in the differentiation state, marked by an increase in the levels of differentiation specific markers K1, involucrin, filaggrin and loricrin while its proliferation status remained unchanged. Rescue experiments with the K5/K14 pair overexpressed in vimentin knockdown background resulted in decreased differentiation state. ΔNp63 emerged as one of the indirect targets of vimentin, through which it modulates the expression levels of K5/K14. Further, immunohistochemistry showed a significant correlation between high vimentin-K14 expression and recurrence/poor survival in oral cancer patients. Thus, in conclusion, vimentin regulates the differentiation switch via modulation of K5/K14 expression. Moreover, vimentin-K14 together may prove to be the novel markers for the prognostication of human oral cancer.</p></div

List of proteins identified using MALDI analysis.

<p>List of proteins identified using MALDI analysis.</p

Schematic representation depicting the role of vimentin in modulation of K5/K14 expression, to regulate differentiation in carcinoma-derived cells.

<p>The model shows the regulation of K5/14 expression by vimentin, perhaps through ΔNp63. Determination of vimentin-K14 status in oral tumor tissues may have clinical implications.</p

High vimentin-K14 expression correlates with poor survival in oral cancer patients.

<p>(A) (a-c) The upper panel show images (Bar: 200μm) of IHC staining for vimentin and K14 expression in cut margin tissues while lower panel (d-f) show images (Bar: 200μm) of IHC staining for vimentin and K14 expression in tumor tissues. The negative control images represent tissue sections incubated with serum from non-immunized mice in place of primary antibodies. (B) The graphical representation shows recurrence with respect to high vimentin-K14 expression and recurrence with respect to the other combinations of vimentin-K14 expression. Kaplan-Meier survival analysis (n = 91) of (C) High vs. low vimentin expression (D) High vs. low K14 expression and (E) High vimentin-K14 vs. the other combinations of vimentin-K14 expression.</p

Downregulation of vimentin resulted in a change in the global keratin profile of the oral SCC derived cell line AW13516.

<p>(A) Schematic representation of the hypothesis. As a tumor cell acquires EMT (marked by upregulation of vimentin), it undergoes transition from a more epithelial-like to a more mesenchymal-like dedifferentiated state. To achieve this transition, there could be a vimentin mediated reprogramming of the keratins which distinguish these states. (B and C) Immunofluorescence (Bar: 10 μm) and western blot analysis of vimentin knockdown (shvim1 and shvim2) and its vector control clone (pTU6vc) using an antibody against vimentin. β-actin was used as the loading control in the western blotting experiment. (D) K8 levels of vimentin knockdown and its vector control clones were analyzed using western blotting. β-actin was used as a loading control. (E) The expression of K8 does not change upon vimentin downregulation. Thus, K8 was used as the loading control for high salt keratin enriched fraction. (F) Representative images of the 2D-gel, which show changes in keratin expression in the high salt keratin enriched fractions of vimentin knockdown and its vector control clones. The black circles indicate similarly expressed while the red circles indicate differentially expressed proteins. All the experiments were repeated independently in triplicates. For all the western blot experiments, the numbers below each blot represent the relative intensity of the bands determined using densitometry.</p

Vimentin knockdown phenotype was partially rescued upon re-expression of ΔNp63α in vimentin knockdown background.

<p>(A) Western blot analysis shows protein levels of ΔNp63 from whole cell lysates of vimentin knockdown and its vector control clones. (B) Fold change in mRNA expression level of ΔNp63 in vimentin knockdown as compared to vector control clones, using qRT-PCR analysis. (C) Western blot analysis shows protein levels of p21 and p27 from the whole cell lysates of vimentin knockdown and its vector control clones. (D) RT-PCR analysis shows expression of ΔNp63α, β and γ isoforms, between vimentin knockdown and vector control clones. GAPDH was used as a loading control. (E) Western blot analysis using anti-flag antibody confirmed the overexpression of flag-tagged ΔNp63α. (F) The protein level of vimentin was tested by western blotting in vimentin knockdown-vector control set and flag-ΔNp63α-vector control set, to confirm the maintenance of vimentin knockdown background in the second group. (G and H) qRT-PCR and western blot analysis of K5 and K14 in flag-ΔNp63α overexpressing and its vector control group. (I) Proliferation curves of flag-ΔNp63α overexpressing and its vector control clones over the period of 4 days, using MTT assay. (J) Representative image of clonogenic assay shows colonies formed by flag-ΔNp63α overexpressing and its vector control clones. (K) QRT-PCR analysis of differentiation specific markers K1, involucrin, filaggrin and loricrin. For all the qRT-PCR experiments, the relative expression of the target gene was normalized to the GAPDH. For all western blotting experiments, β-actin was used as a loading control. The graphical data represents ± SEM of three independent experiments.</p

Vimentin mediated positive regulation of K5/K14 levels is not a cell line specific phenomenon.

<p>(A and B) Western blot analysis shows protein level of vimentin, K5, K14, K17 and involucrin in vimentin overexpressing clones of A431vim and HaCatvim as compared to its respective vector control clones A431vc and HaCatvc. Since A431 does not express K14, whole cell lysate from AW13516 was used as a positive control. (C) Whole cell lysates from DOK, AW13516 and AW8507 cells were probed with antibodies against vimentin, K5, K14 and involucrin respectively using western blotting. (D and E) Western blot and RT-PCR analysis of K14 and vimentin in K14 knockdown (shRK14-K7 and shRK14-K9) and its vector control clones (pTU6-AW1). GAPDH was used as the loading control in RT-PCR experiment. β-actin was used as the loading control in western blotting experiments. Western blotting experiments were done thrice with three independent sets of samples.</p

Phenotypic alterations associated with K5/K14 downregulation as a consequence of vimentin depletion.

<p>(A) Proliferation curves of vimentin knockdown and its vector control clones over a period of 4 days, using MTT assay. (B) Representative image of the clonogenic assay shows colonies formed by vimentin knockdown and its vector control clones. (C) qRT-PCR analysis of differentiation specific markers K1, involucrin, filaggrin and loricrin respectively. The relative expression of the target gene was normalized to GAPDH. For (A and C), the graphical data represents ± SEM of three independent experiments. (D) Representative images of tumorigenicity assays using NOD-SCID mice (6 animals each) injected with either vimentin knockdown or vector control clones. The tumors are indicated by dotted circles. (E) The tumor measurements were recorded up to 42 days, after which the animals were sacrificed and the tumor tissue was isolated for IHC staining. The graph shows the tumor volume plotted against time for both the clones. (F) Representative images (Bar: 200μm) of IHC staining for expression of vimentin, K5 and K14 respectively in mice tumor tissues. The negative control images represent tissue sections incubated with serum from non-immunized mice in place of primary antibodies.</p