Active heat shock transcription factor 1 supports migration of the melanoma cells via vinculin down-regulation

AbstractHeat shock transcription factor 1 (HSF1), the major regulator of stress response, is frequently activated in cancer and has an apparent role in malignant transformation. Here we analyzed the influence of the over-expression of a constitutively active transcriptionally-competent HSF1 mutant form on phenotypes of mouse and human melanoma cells. We observed that the expression of active HSF1 supported anchorage-independent growth in vitro, and metastatic spread in the animal model in vivo, although the proliferation rate of cancer cells was not affected. Furthermore, active HSF1 enhanced cell motility, reduced the adherence of cells to a fibronectin-coated surface, and affected the actin cytoskeleton. We found that although the expression of active HSF1 did not affect levels of epithelial-to-mesenchymal transition markers, it caused transcriptional down-regulation of vinculin, protein involved in cell motility, and adherence. Functional HSF1-binding sites were found in mouse and human Vcl/VCL genes, indicating a direct role of HSF1 in the regulation of this gene. An apparent association between HSF1-induced down-regulation of vinculin, increased motility, and a reduced adherence of cells suggests a possible mechanism of HSF1-mediated enhancement of the metastatic potential of cancer cells

PHLDA1 Does Not Contribute Directly to Heat Shock-Induced Apoptosis of Spermatocytes

Spermatocytes are among the most heat-sensitive cells and the exposure of testes to heat shock results in their Heat Shock Factor 1 (HSF1)-mediated apoptosis. Several lines of evidence suggest that pleckstrin-homology-like domain family A, member 1 (PHLDA1) plays a role in promoting heat shock-induced cell death in spermatogenic cells, yet its precise physiological role is not well understood. Aiming to elucidate the hypothetical role of PHLDA1 in HSF1-mediated apoptosis of spermatogenic cells we characterized its expression in mouse testes during normal development and after heat shock. We stated that transcription of Phlda1 is upregulated by heat shock in many adult mouse organs including the testes. Analyzes of the Phlda1 expression during postnatal development indicate that it is expressed in pre-meiotic or somatic cells of the testis. It starts to be transcribed much earlier than spermatocytes are fully developed and its transcripts and protein products do not accumulate further in the later stages. Moreover, neither heat shock nor expression of constitutively active HSF1 results in the accumulation of PHLDA1 protein in meiotic and post-meiotic cells although both conditions induce massive apoptosis of spermatocytes. Furthermore, the overexpression of PHLDA1 in NIH3T3 cells leads to cell detachment, yet classical apoptosis is not observed. Therefore, our findings indicate that PHLDA1 cannot directly contribute to the heat-induced apoptosis of spermatocytes. Instead, PHLDA1 could hypothetically participate in death of spermatocytes indirectly via activation of changes in the somatic or pre-meiotic cells present in the testes

Crosstalk between HSF1 and HSF2 during the heat shock response in mouse testes

Heat Shock Factor 1 (HSF1) is the primary transcription factor responsible for the response to cellularstress, while HSF2 becomes activated during development and differentiation, including spermatogen-esis. Although both factors are indispensable for proper spermatogenesis, activation of HSF1 by heatshock initiates apoptosis of spermatogenic cells leading to infertility of males. To characterize mecha-nisms assisting such heat induced apoptosis we studied how HSF1 and HSF2 cooperate during the heatshock response. For this purpose we used chromatin immunoprecipitation and the proximity ligationapproaches. We looked for co-occupation of binding sites by HSF1 and HSF2 in untreated (32◦C) or heatshocked (at 38◦C or 43◦C) spermatocytes, which are cells the most sensitive to hyperthermia. At thephysiological temperature or after mild hyperthermia at 38◦C, the sharing of binding sites for both HSFswas observed mainly in promoters of Hsp genes and other stress-related genes. Strong hyperthermiaat 43◦C resulted in an increased binding of HSF1 and releasing of HSF2, hence co-occupation of pro-moter regions was not detected any more. The close proximity of HSF1 and HSF2 (and/or existence ofHSF1/HSF2 complexes) was frequent at the physiological temperature. Temperature elevation resultedin a decreased number of such complexes and they were barely detected after strong hyperthermia at43◦C. We have concluded that at the physiological temperature HSF1 and HSF2 cooperate in spermato-genic cells. However, temperature elevation causes remodeling of chromatin binding and interactionsbetween HSFs are disrupted. This potentially affects the regulation of stress response and contributes tothe heat sensitivity of these cells

BRAFV600E-Associated Gene Expression Profile: Early Changes in the Transcriptome, Based on a Transgenic Mouse Model of Papillary Thyroid Carcinoma

<div><p>Background</p><p>The molecular mechanisms driving the papillary thyroid carcinoma (PTC) are still poorly understood. The most frequent genetic alteration in PTC is the <i>BRAF</i>V600E mutation–its impact may extend even beyond PTC genomic profile and influence the tumor characteristics and even clinical behavior.</p><p>Methods</p><p>In order to identify <i>BRAF</i>-dependent signature of early carcinogenesis in PTC, a transgenic mouse model with <i>BRAF</i>V600E-induced PTC was developed. Mice thyroid samples were used in microarray analysis and the data were referred to a human thyroid dataset.</p><p>Results</p><p>Most of <i>BRAF</i>(+) mice developed malignant lesions. Nevertheless, 16% of <i>BRAF</i>(+) mice displayed only benign hyperplastic lesions or apparently asymptomatic thyroids. After comparison of non-malignant <i>BRAF</i>(+) thyroids to <i>BRAF</i>(−) ones, we selected 862 significantly deregulated genes. When the mouse <i>BRAF</i>-dependent signature was transposed to the human HG-U133A microarray, we identified 532 genes, potentially indicating the <i>BRAF</i> signature (representing early changes, not related to developed malignant tumor). Comparing <i>BRAF</i>(+) PTCs to healthy human thyroids, PTCs without <i>BRAF</i> and <i>RET</i> alterations and <i>RET</i>(+), <i>RAS</i>(+) PTCs, 18 of these 532 genes displayed significantly deregulated expression in all subgroups. All 18 genes, among them 7 novel and previously not reported, were validated as <i>BRAF</i>V600E-specific in the dataset of independent PTC samples, made available by The Cancer Genome Atlas Project.</p><p>Conclusion</p><p>The study identified 7 <i>BRAF</i>-induced genes that are specific for <i>BRAF V600E</i>-driven PTC and not previously reported as related to <i>BRAF</i> mutation or thyroid carcinoma: <i>MMD</i>, <i>ITPR3</i>, <i>AACS</i>, <i>LAD1</i>, <i>PVRL3</i>, <i>ALDH3B1</i>, and <i>RASA1</i>. The full signature of <i>BRAF</i>-related 532 genes may encompass other <i>BRAF</i>-related important transcripts and require further study.</p></div

Microarray-derived dataset- human cohort.

<p>PTC(-)- PTCs without any mutation detected</p><p>Microarray-derived dataset- human cohort.</p

Hierarchical clustering of mouse samples.

<p>Thirty-eight mouse samples based on 1020 probe sets significantly differentiating between <i>BRAF</i>(+) and <i>BRAF</i>(−) non-malignant mouse samples (marked with blue and cyan respectively). PTCs (red); borderline thyroid lesions (BL; magenta); benign hyperplastic thyroid lesions (BHL; dark green); asymptomatic thyroid glands (AT; green).</p

Boxplots of 18 genes chosen for validation.

<p>Expression distribution for each gene from our microarray data (on the left), The Cancer Genome Atlas Project data (on the right). The expression levels of analyzed genes are presented in <i>BRAF</i>(+) PTCs, <i>RET</i>(+), <i>RAS</i>(+), PTC(-) and healthy thyroids (HT) from left to right, respectively (as presented at the bottom of the figure). FDR values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143688#pone.0143688.t004" target="_blank">Table 4</a>.</p

Pathway analysis of the 532-gene signature of BRAFV600E-induced PTC early carcinogenesis.

<p>Pathway analysis of the 532-gene signature of BRAFV600E-induced PTC early carcinogenesis.</p