HSP110 promotes colorectal cancer growth through STAT3 activation.

Heat shock protein 110 (HSP110) is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps cells survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently shown that colorectal cancer patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110-inactivating mutation (HSP110DE9). In this work, we used patient biopsies, human colorectal cancer cells grown in vitro and in vivo (xenografts), and intestinal crypts to demonstrate that HSP110 is also involved in colon cancer growth. We showed that HSP110 induces colon cancer cell proliferation and that this effect is associated with STAT3 activation, specifically an increase in STAT3 phosphorylation, nuclear translocation and transcription factor activity. STAT3 inhibition blocks the proliferative effect of HSP110. From a molecular standpoint, we demonstrated that HSP110 directly binds to STAT3, thereby facilitating its phosphorylation by JAK2. Finally, we showed a correlation between HSP110 expression and STAT3 phosphorylation in colon cancer patient samples. Thus, the expression of HSP110 in colon cancer contributes to STAT3-dependent tumor growth and the frequent inactivating mutation of this chaperone is probably an important event underlying the improved prognosis in colon cancer displaying MSI.

Dual regulation of SPI1/PU.1 transcription factor by heat shock factor 1 (HSF1) during macrophage differentiation of monocytes.

In addition to their cytoprotective role in stressful conditions, heat shock proteins (HSPs) are involved in specific differentiation pathways, for example, we have identified a role for HSP90 in macrophage differentiation of human peripheral blood monocytes that are exposed to macrophage colony-stimulating factor (M-CSF). Here, we show that deletion of the main transcription factor involved in heat shock gene regulation, heat shock factor 1 (HSF1), affects M-CSF-driven differentiation of mouse bone marrow cells. HSF1 transiently accumulates in the nucleus of human monocytes undergoing macrophage differentiation, including M-CSF-treated peripheral blood monocytes and phorbol ester-treated THP1 cells. We demonstrate that HSF1 has a dual effect on SPI1/PU.1, a transcription factor essential for macrophage differentiation and whose deregulation can lead to the development of leukemias and lymphomas. Firstly, HSF1 regulates SPI1/PU.1 gene expression through its binding to a heat shock element within the intron 2 of this gene. Furthermore, downregulation or inhibition of HSF1 impaired both SPI1/PU.1-targeted gene transcription and macrophage differentiation. Secondly, HSF1 induces the expression of HSP70 that interacts with SPI1/PU.1 to protect the transcription factor from proteasomal degradation. Taken together, HSF1 appears as a fine-tuning regulator of SPI1/PU.1 expression at the transcriptional and post-translational levels during macrophage differentiation of monocytes.

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells.

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E.

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

Heat shock protein 27 is involved in SUMO-2/3 modification of heat shock factor 1 and thereby modulates the transcription factor activity.

Heat shock protein 27 (HSP27) accumulates in stressed cells and helps them to survive adverse conditions. We have already shown that HSP27 has a function in the ubiquitination process that is modulated by its oligomerization/phosphorylation status. Here, we show that HSP27 is also involved in protein sumoylation, a ubiquitination-related process. HSP27 increases the number of cell proteins modified by small ubiquitin-like modifier (SUMO)-2/3 but this effect shows some selectivity as it neither affects all proteins nor concerns SUMO-1. Moreover, no such alteration in SUMO-2/3 conjugation is achievable by another HSP, such as HSP70. Heat shock factor 1 (HSF1), a transcription factor responsible for HSP expression, is one of the targets of HSP27. In stressed cells, HSP27 enters the nucleus and, in the form of large oligomers, binds to HSF1 and induces its modification by SUMO-2/3 on lysine 298. HSP27-induced HSF1 modification by SUMO-2/3 takes place downstream of the transcription factor phosphorylation on S303 and S307 and does not affect its DNA-binding ability. In contrast, this modification blocks HSF1 transactivation capacity. These data show that HSP27 exerts a feedback inhibition of HSF1 transactivation and enlighten the strictly regulated interplay between HSPs and HSF1. As we also show that HSP27 binds to the SUMO-E2-conjugating enzyme, Ubc9, our study raises the possibility that HSP27 may act as a SUMO-E3 ligase specific for SUMO-2/3.

TNFalpha stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells.

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.

Regulation of death receptors--relevance in cancer therapies.

Apoptosis is an essential non-inflammatory mechanism for cell removal, which occurs during both physiological and pathological conditions. Apoptosis is characteristically executed by cysteine proteases, termed caspases. The most specific way to activate the caspases machinery is through death receptors (DRs), such as the tumor necrosis factor (TNFR), Fas receptor (FasR), and TRAIL (TRAIL-R). The apoptotic signaling is tightly regulated by the balance of pro-apoptotic and anti-apoptotic proteins and an imbalance between cell death and proliferation may cause numerous diseases, including cancers. The intensive research during the past decade has delineated the basic mechanisms of apoptosis and outlined many important molecular mechanisms underlying the regulation of apoptosis. There is also a better understanding of how the regulation of apoptosis can be disturbed in human cancer cells. The interplay between DRs signaling and anticancer drugs has offered new concepts for the development of highly specific therapeutical agents. Here we review the current understanding of the different molecular mechanisms that regulate DR-mediated apoptosis and the defects in apoptotic signaling discovered in cancer cells. In light of this knowledge, new promising target-based agents for future cancer therapies have been developed.

Role of protein kinase C zeta isoform in Fas resistance of immature myeloid KG1a leukemic cells.

Leukemic CD34(+) immature acute myeloid leukemia (AML) cells express Fas receptor but are frequently resistant to Fas agonistic reagents. Fas plays an important role in T-cell-mediated cytotoxicity, and recently it has been suggested that altered Fas signaling may contribute to drug resistance. Therefore, Fas resistance could be one of the mechanisms by which AML progenitors escape chemotherapy or T-cell-based immune intervention. However, the molecular mechanism of Fas resistance in AML cells has not been identified. Fas signaling can be interrupted at 3 mains levels: Fas clustering, alteration of death-inducing-signaling-complex (DISC) formation, and effector caspase inhibition of downstream caspase-8. This study shows that in the Fas-resistant CD34(+)CD38(-) KG1a cells, Fas agonists resulted in Fas aggregation but not in caspase-8 activation, related to a defect in DISC formation. However, pretreatment with chelerythrin, but not with calphostin C, resulted in the restoration of Fas-induced caspase-8 activation and cytotoxicity, suggesting that some atypical protein kinase C (PKC) isoforms contributed to the lack of DISC formation. Indeed, treatment with antisense oligonucleotides directed against PKC zeta and enforced expression of Par-4, a negative regulator of PKC zeta activity, restored Fas-induced caspase-8 activity and apoptosis. Moreover, it was found that PKC zeta interacts with FADD and that PKC zeta immunoextracts prepared from KG1a cells are able to phosphorylate FADD in vitro, whereas this phosphorylation is dramatically reduced in Par-4 transfectant cells. In conclusion, it is suggested that in AML cells, PKC zeta plays an important role in Fas resistance by inhibiting DISC formation, possibly by phosphorylating FADD.