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The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

ABSTRACT: It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

SUBMITTER: Lorenzo-Martin LF

PROVIDER: S-EPMC9187518 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

Lorenzo-Martín L Francisco LF Menacho-Márquez Mauricio M Fernández-Parejo Natalia N Rodríguez-Fdez Sonia S Pascual Gloria G Abad Antonio A Crespo Piero P Dosil Mercedes M Benitah Salvador A SA Bustelo Xosé R XR

Oncogene 20220509 24

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate ...[more]

PMID: 35534539

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Project description:The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, and many of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs. Microarray analyses revealed that Vav2 and Vav3 controlled a vast transcriptional program in breast cancer cells through mechanisms that were shared between the two proteins, isoform-specific or synergistic. Furthermore, the abundance of Vav regulated transcripts was modulated by Rac1-dependent and Rac1-independent pathways. This transcriptome encoded therapeutically targetable proteins that played non redundant roles in primary tumorigenesis and lung-specific metastasis, such as integrin-linked kinase (Ilk), the transforming growth factorM-bM-^@M-^Sb family ligand inhibin bA, cyclooxygenase-2, and the epithelial cell adhesion molecule Tacstd2. It also contained gene signatures that predicted disease outcome in breast cancer patients. These results identify possible targets for treating breast cancer and lung metastases and provide a potential diagnostic tool for clinical use. All microarray experiments were performed by the personnel of the Genomics and Proteomics Unit of our Institution. Transcriptomal changes were determined using the Mouse Gene 1.0 ST arrays (Affymetrix). Two independent experiments were performed to identify the Vav2/Vav3-dependent transcriptome. In the first one, we compared the transcriptomes of Control, KD2/3(A) and KD2/3(B) to identify Vav family-dependent genes. In the second one, we compared the transcriptomes of KD2/3(A) and KD2/3+V2/3 cells. In all cases, total cellular RNA was extracted from three independent exponential cultures of the appropriate cell lines using the RNAeasy kit (Qiagen), quantified using 6000 Nano Chips (Agilent Technologies, Santa Clara, CA), and used (2.3 M-NM-<g/sample) to generated labeled cRNA probes according to the manufacturerM-bM-^@M-^Ys instructions (Affymetrix). Upon microarray hybridization, the raw data was normalized, filtered and analyzed with the Bioconductor software (www.bioconductor.com) using the Affy and Siggenes applications. Generation of knockdown cell lines. The shRNA-mediated knockdown of transcripts for Vav2 and Vav3 was carried out using already packaged Mission TRC lentiviral particles (Sigma-Aldrich) according to the manufacturerM-bM-^@M-^Ys protocol. The catalogue numbers and shRNA sequences yielding the greatest knockdown were clone number TRC0000097094 (5M-bM-^@M-^Y-CCGGGCCTGCATCTCTGGTTTAGATCTCGAGATCTAA ACCAGAGATGCAGGCTTTTTG-3M-bM-^@M-^Y) for the mouse Vav2 mRNA; TRC0000097124 (5M-bM-^@M-^Y-CCGGCCAGCATTTCTCGTCTTAAATCTCGAGATTTAA GACGAGAAATGCTGGTTTTTG-3M-bM-^@M-^Y) for the mouse Vav3 mRNA. Lentiviral particles containing the empty pLOK.1puro vector (Sigma-Aldrich) were used to generate the M-bM-^@M-^\ControlM-bM-^@M-^] 4T1 cells used in these experiments. Cells were incubated with lentiviral particles in the presence of 8 M-NM-<g/ml polybrene (Sigma), selected with puromycin (1 M-NM-<g/ml; Sigma), and used as either pools or isolated clones. Two clones of double Vav2;Vav3 knockdown cells (designated KD2/A(A) and KD2/3 (B)) were used in these experiments. Generation of rescued 4T1 cell lines. To generate the rescued KD2/3(A) cell line expressing both Vav2 and Vav3, cells were first infected with lentiviral particles produced from the pCCM33 vector (encoding wild type, HA-tagged Vav2) and then with lentiviral particles containing the pCQS1 vector (encoding wild type, Myc-tagged Vav3). Cells were then selected with hygromycin (present in the Vav2-encoding pCCM33 vector) and susbsequently sorted by flow cytometry to isolate GFP positive cells (which was expressed bicistronically from the same Vav3-encoding pCQS1 vector). A clone of double reconstituted cells was used in the microarray experiments and designated as KD2/3+V2/V3.

Dataset Information

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

Publications

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

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