The small heat shock protein Hsp27 is expressed at high levels in many tumors and provides protection against anticancer drugs. Here, we show that expression of

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45°C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72⌬EEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2915 KEY WORDS HSF1, heat shock proteins, migration, ERK, JNK ABBREVIATIONS ABSTRACTUpon cancer progression in mouse models of prostate cancer, the heat shock transcription factor Hsf1 becomes strongly upregulated, especially in metastases. We hypothesized that Hsf1 plays a role in cell migration, a process necessary for metastases. Using a cell culture model of migration in a scratch, we found that immortalized MEF cells derived from hsf1 -/-animals were deficient in both basal and EGF-induced migration. MEF cell migration was dependent on JNK and ERK signaling, since inhibition of these pathways blocked EGF-stimulated cell migration. ERK was activated at the edge of the scratch in parental cells, and this activity was further increased after addition of EGF. Both basal and EGF-stimulated ERK activation were suppressed in hsf1 -/-cells at the edge of the scratch. Furthermore, activation of ERK and JNK pathways by EGF was reduced in hsf1 -/-cells. The impairment of MAP kinase signaling in hsf1 -/-cells was partly due to the reduced expression of EGFR1. In addition, knockout of Hsf1 gene caused a second defect in MAP kinase signaling probably at the level of Ras. We conclude that HSF1 is necessary for MAP kinase signaling which in turn affects the EGF-induced cell migration.

Depletion of the major heat shock protein Hsp72 leads to activation of the senescence program in a variety of tumor cell lines via both p53-dependent and p53-independent pathways. Here, we found that the Hsp72-depleted cells show defect in phosphorylation and activation of the protein kinase Chk1 by genotoxic stresses, such as UVC irradiation or camptothecin. Under these conditions, phosphorylation of Rad17 was also suppressed, whereas phosphorylation of p53 at Ser 15 was not affected, indicating a specific defect in phosphorylation of a subset of the ATR kinase substrates. Similarly, suppression of Chk1 activation was seen when senescence signaling was triggered by direct stimulation of p53, depletion of Cdc2, or overexpression of the cell cycle inhibitors p21 or p16. Thus, defect in Chk1 activation was not a consequence of the chaperone imbalance, but rather a downstream effect of activation of the senescence signaling. Inhibition of Chk1 was associated with inefficient inter-S phase checkpoint, as Hsp72 depleted cells failed to halt cell cycle progression upon UVC irradiation. Accordingly, sensitivity of cells to genotoxic stimuli after Hsp72 depletion was significantly enhanced. Thus, activation of the senescence signaling causes a defect in the DNA damage response manifested in increased sensitivity to genotoxic stresses.

Abstract:Anti-apoptotic functions of heat shock proteins Hsp70 and Hsp27 are well established. However, radiation and genotoxic antineoplastic drugs at clinically relevant doses induce apoptosis mostly in lymphoid cells, while in epithelial tumors they evoke different type of response, mainly senescence and mitotic catastrophe, which leads to loss of clonogenic potential of cells. Here we review old and new data showing that upregulation of Hsp27 or Hsp70 levels protect various tumor cell lines from gamma-and UV-radiation and genotoxic anti-neoplastic drugs. Accordingly, downregulation of Hsp27 or Hsp70 levels by antisense or siRNA sensitizes tumor cells to these agents. Importantly, protection and sensitization by modulation of Hsp27 or Hsp70 levels were manifested not only by modulation of apoptosis, but by clonogenic survival as well, and recent data indicate that these Hsps can suppress also drug-induced senescence. Several studies demonstrated that intrinsic and acquired chemo-and radioresistance in tumor cell lines and in patients with certain forms of cancers can be associated with upregulation of Hsp27 and/or Hsp70. Possible mechanisms of Hsp-induced protection, in particular, modulation of p53-dependent and p53-independent DNA-damaging signaling pathways, are discussed