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. 2023 Dec 26;42(12):113470.
doi: 10.1016/j.celrep.2023.113470. Epub 2023 Nov 20.

Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas

Nick A Kuburich  1 , Petra den Hollander  1 , Maria Castaneda  2 , Mika Pietilä  3 , Ximing Tang  2 , Harsh Batra  2 , Francisco Martínez-Peña  4 , Tanvi H Visal  2 , Tieling Zhou  2 , Breanna R Demestichas  1 , Ritesh V Dontula  2 , Jojo Y Liu  2 , Joanna Joyce Maddela  1 , Reethi S Padmanabhan  2 , Lan Thi Hanh Phi  2 , Matthew J Rosolen  2 , Thiru Sabapathy  1 , Dhiraj Kumar  5 , Filippo G Giancotti  5 , Luke L Lairson  4 , Maria Gabriela Raso  2 , Rama Soundararajan  2 , Sendurai A Mani  6
Affiliations

Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas

Nick A Kuburich et al. Cell Rep. .

Abstract

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.

Keywords: CP: Cancer; cancer stem cells, epithelial-mesenchymal transition, CSC, EMT, multinucleation, vimentin, metastasis, stemness, hybrid E/M, breast cancer.

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Conflict of interest statement

Declaration of interests The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. Hybrid E/M cancer cells have stemness properties and become multinucleated when vimentin phosphorylation is stabilized, whereas epithelial and mesenchymal cells do not
(A) Western blot analysis of epithelial and mesenchymal markers in indicated cell lines, which were classified as epithelial (blue), hybrid E/M (orange), or mesenchymal (purple) based on these markers. (B) Left: Mean number of mammospheres formed by indicated cell lines. Right: Violin plot of numbers of mammospheres formed by epithelial (E), hybrid E/M (E/M), and mesenchymal (M) lines with values for individual lines shown as black dots. (C) Left: Percentages of relative multinucleated cells in the samples treated with 0.5 μM FiVe1 versus samples treated with 0.1% DMSO. Right: Violin plot of the relative multinucleation in E, E/M, and M lines. Relative percent multinucleation was calculated by dividing the percentage of multinucleated cells in FiVe1-treated samples by the percentage of multinucleated cells in vehicle-treated samples. (D) Western blot analysis of vimentin-S56 phosphorylation (VIM-pS56) and total vimentin (VIM) in indicated cells following treatment with vehicle (−) or FiVe1 (+). An unpaired t test was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. NS, not significant. Error bars are ± SEM.
Figure 2.
Figure 2.. Stabilization of vimentin-S56 phosphorylation in CSC-enriched populations increases multinucleation and inhibits SSCDs
(A) Schematics showing monolayer culture (top) and suspension culture, which is CSC enriched (bottom) before cells were disassociated, plated, and treated with 2 μg/mL doxycycline or 0.5 μM FiVe1. Image created with BioRender.com. (B) Relative percentages of multinucleated 4T1 VIM-S56E cells treated with doxycycline following monolayer or suspension growth. (C) Relative percentages of multinucleated 4T1, SUM159, and MDA-MB-231 cells treated with FiVe1 following monolayer or suspension growth. (D) Representative fluorescence-activated cell sorting analysis of HMLER and HCC1806 cells gated for CD44high/CD24low and CD44low/CD24high populations. (E) Relative percentages of multinucleation of CD44high/CD24low and CD44low/CD24high HMLER (left) and CD44high/CD24low and CD44low/CD24high HCC1806 (right) cells treated with FiVe1. (F) Left: Representative images of FOXC2 and Numb immunofluorescence staining of 4T1 cells showing staining patterns of cells undergoing symmetrical differentiated cell division (SDCD) and SSCD. Right: Percentages of cells undergoing SDCD, SSCD, and asymmetrical stem cell division (ASCD) in 4T1 cultures treated with 0.1% DMSO or 1 μM FiVe1. (G) Left: Representative images of FOXC2 and Numb immunofluorescence staining of SUM159 cells showing staining patterns of cells undergoing SDCD and SSCD. Right: Percentages of cells undergoing SDCD, SSCD, and ASCD in SUM159 cultures treated with 0.1% DMSO or 1.2 μM FiVe1. An unpaired t test was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001; NS indicates not significant. Error bars are ± SEM.
Figure 3.
Figure 3.. Removing multinucleated cells reduces the stemness properties and metastatic potential of tumor cells
(A) Schematic showing the isolation of 2N (non-multinucleated) cells by fluorescence-activated cell sorting (FACS) based on DNA content following treatment with 0.1% DMSO or 1 μM FiVe1. The isolated 2N cells were then used in downstream assays. Image created with BioRender.com. (B) Left: Representative images of mammosphere colonies of 4T1 2N sorted cells after the indicated treatment with DMSO or FiVe1. Scale bars, 100 μm. Right: Numbers of mammospheres formed from 4T1 2N cells treated with vehicle or FiVe1. (C) Photographs of all tumors isolated from the limiting dilution tumor initiation assay. Units on the ruler are in centimeters. (D) Table of frequencies of tumor formation per fat pad injected with 2N cells treated with vehicle or FiVe1. (E) The average volume of primary tumors formed using 2N cells from the DMSO- or FiVe1-treated culture treatment at the end of the experiment. (F) Left: Representative images of lungs for 4T1 2C sorted cells after the indicated pretreatment conditions spontaneous metastasis experiment. Black arrows indicate visible lung nodules. Right: Bar graph showing the average number of visible lung nodules for the indicated conditions. (G) Representative images of H&E-stained lungs from 2C sorted 4T1 spontaneous metastasis experiment. Scale bars, 1 mm. Relative metastatic burden was calculated by measuring the area of the entire lung and then measuring the area of metastasis. The metastatic area was divided by the total lung area and multiplied by 100. An unpaired t test was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Error bars are ± SEM.
Figure 4.
Figure 4.. A phosphomimetic mutation at S56 in vimentin or stabilization of the phosphorylation of S56 with FiVe1 decreases spontaneous metastasis
(A) Mean volume of 4T1 TET-on VIM-S56E primary tumors at the experimental endpoint in mice given sucrose-water vehicle control or doxycycline. (B) Left: Representative images of lungs from mice administered water or Dox. Black arrows indicate lung nodules. Right: Average numbers of visible lung nodules in indicated conditions. (C) Representative images of H&E-stained lungs. Scale bars, 1 mm. Relative metastatic burden was calculated by dividing the metastatic area by the area of the entire lung and multiplying by 100. (D) Average volume of 4T1 primary tumors at the experimental endpoint in mice injected with 4T1 cells pretreated with vehicle or FiVe1. (E) Left: Representative images of lungs from mice injected with 4T1 cells pretreated with vehicle or FiVe1. Black arrows indicate lung nodules. Right: Average numbers of visible lung nodules. (F) Representative images of H&E-stained lungs. Scale bars, 1 mm. Relative metastatic burden was calculated by dividing the metastatic area by the area of the entire lung and multiplying by 100. An unpaired t test was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. NS indicates not significant. Error bars are ± SEM.
Figure 5.
Figure 5.. Experimental metastasis is inhibited following stabilizing vimentin-S56 phosphorylation
(A) Left: Representative images of lungs from mice injected with 4T1 TET-on VIM-S56E cells and given sucrose-water or doxycycline in sucrose-water. Black arrows indicate visible lung nodules. Right: Average numbers of visible lung nodules. (B) Representative images of H&E-stained lungs from 4T1 TET-on VIM-S56E experimental metastasis experiment. Scale bars, 1 mm. Relative metastatic burden was calculated by dividing the metastatic area by the area of the entire lung and multiplying by 100. (C) Left: Representative images of lungs from mice injected with 4T1 cells treated with 0.1% DMSO or 1 μM FiVe1. Black arrows indicate visible lung nodules. Right: Average numbers of visible lung nodules. (D) Representative images of H&E-stained lungs. Scale bars, 1 mm. Relative metastatic burden was calculated by dividing the metastatic area by the area of the entire lung and multiplying by 100. An unpaired t test was used to calculate significance. *p < 0.05; **p < 0.01. Error bars are ± SEM.
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