Heat shock-induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function

TAR DNA-binding protein (TDP-43) is a highly conserved and essential DNA- and RNA-binding protein that controls gene expression through RNA processing, in particular, regulation of splicing. Intracellular aggregation of TDP-43 is a hallmark of amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. This TDP-43 pathology is also present in other types of neurodegeneration including Alzheimer's disease. We report here that TDP-43 is a substrate of MEK, a central kinase in the MAPK/ERK signaling pathway. TDP-43 dual phosphorylation by MEK, at threonine 153 and tyrosine 155 (p-T153/Y155), was dramatically increased by the heat shock response (HSR) in human cells. HSR promotes cell survival under proteotoxic conditions by maintaining protein homeostasis and preventing protein misfolding. MEK is activated by HSR and contributes to the regulation of proteome stability. Phosphorylated TDP-43 was not associated with TDP-43 aggregation, and p-T153/Y155 remained soluble under conditions that promote protein misfolding. We found that active MEK significantly alters TDP-43-regulated splicing and that phosphomimetic substitutions at these two residues reduce binding to GU-rich RNA. Cellular imaging using a phospho-specific p-T153/Y155 antibody showed that phosphorylated TDP-43 was specifically recruited to the nucleoli, suggesting that p-T153/Y155 regulates a previously unappreciated function of TDP-43 in the processing of nucleolar-associated RNA. These findings highlight a new mechanism that regulates TDP-43 function and homeostasis through phosphorylation and, therefore, may contribute to the development of strategies to prevent TDP-43 aggregation and to uncover previously unexplored roles of TDP-43 in cell metabolism

Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer

Pancreatic ductal adenocarcinoma is a lethal disease with limited treatment options and poor survival. We studied 83 spatial samples from 31 patients (11 treatment-naïve and 20 treated) using single-cell/nucleus RNA sequencing, bulk-proteogenomics, spatial transcriptomics and cellular imaging. Subpopulations of tumor cells exhibited signatures of proliferation, KRAS signaling, cell stress and epithelial-to-mesenchymal transition. Mapping mutations and copy number events distinguished tumor populations from normal and transitional cells, including acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Pathology-assisted deconvolution of spatial transcriptomic data identified tumor and transitional subpopulations with distinct histological features. We showed coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin in tumor cells. Chemo-resistant samples contain a threefold enrichment of inflammatory cancer-associated fibroblasts that upregulate metallothioneins. Our study reveals a deeper understanding of the intricate substructure of pancreatic ductal adenocarcinoma tumors that could help improve therapy for patients with this disease

Characterization of human follicular thyroid cancer cell lines in preclinical mouse models

Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2) with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing

Phenotypic characterization of metastatic anaplastic thyroid cancer stem cells.

Emerging evidence suggests cancer stem cells (CSCs) may initiate new tumors in anaplastic thyroid carcinoma (ATC), one of the most aggressive solid tumors in humans. However, the involvement of CSCs in human tumorigenesis has not been previously studied in authenticated ATC cell lines. Here we demonstrate a functional role of CSCs in four new validated human ATC cell lines (THJ-11T, THJ-16T, THJ-21T and THJ-29T). We identified and enriched CSCs using a spheroid-forming assay. About 3 to 9% of cells from four ATC cell lines formed thyrospheres. The thyrospheres expressed the stem cell markers NANOG and Oct4 and possessed the ability to self-renew. Injection of these thyrospheres into the thyroids of NOD/SCID Il2rg-/- mice resulted in the formation of metastatic tumors that recapitulated the clinical features of human ATC. To our knowledge, this is the first in vivo characterization of thyroid CSCs using validated human ATC cell lines. The availability of disease-specific thyrospheres and our orthotopic tumor models will enable the elucidation of disease mechanisms and the environmental niche of CSCs. They may also be useful for preclinical therapeutic screening and for monitoring the effects of biological therapies on ATC

The effect of cisplatin on ATC cells.

<p>(A) Representative phase contrast microscopy images of THJ-11T parental monolayer-derived cells exposed to the indicated dose of cisplatin for 48 hours. (B) Dose-dependent inhibition of growth in all ATC cell lines. IC₅₀ values are as shown for each cell line. (C) Comparison of <i>in vitro</i> resistance to cisplatin of the parental monolayer cells and spheroid-forming cells. The THJ-11T and THJ-16T parental monolayer-derived cells and thyrosphere-derived cells were treated with 10 µM of cisplatin and the fraction of proliferating cells remaining was subsequently estimated based on the Alamar Blue cell proliferation assay. All experiments were performed in triplicate. ****, <i>P</i><0.0001; ***, <i>P</i><0.001; **, <i>P</i><0.05.</p

Limiting dilution analysis using human ATC cell lines in a subcutaneous mouse model of thyroid carcinoma.

<p>Limiting dilution analysis using human ATC cell lines in a subcutaneous mouse model of thyroid carcinoma.</p

ATC monolayer-derived cells initiate tumors in serially transplanted immunodeficient mice.

<p>(A) Tumor growth curve generated by subcutaneous injection of THJ-11T parental monolayer-derived cells. The number of cells injected is indicated. (B) A representative subcutaneous tumor removed from a mouse xenograft. (C) Western blot analysis showing the expression of ALDH, CD44, and CXCR4 in cells derived from primary, secondary and tertiary xenografts. (D) H&E staining showed that tumors originating from primary, secondary and tertiary xenografts are composed of a highly heterogeneous population of cells with a large nucleus-to-cytoplasm ratio. (E) Immunohistochemical analysis of ALDH, CD44, CXCR4 and CD133 levels in mouse xenografts generated from primary xenografts of THJ-11T and THJ-16T. Note that mouse xenografts do not express CD133.</p

Characterization of ATC cells <i>in vitro</i>.

<p>(A) Phase contrast microscopy images of four ATC cell lines cultured as monolayers in RPMI medium. (B) Growth proliferation curve over 96 hours demonstrates the proliferative potential of these ATC cell lines. (C) qRT-PCT analysis of thyroid transcription factors <i>Pax8</i> and <i>TTF1</i> and thyroid differentiation markers <i>TSHR</i>, <i>TG</i>, <i>NIS</i>, and <i>TPO</i>. Human GAPDH was used as the housekeeping gene during the amplifications.</p