Distinct effects of heat shock temperatures on mitotic progression by influencing the spindle assembly checkpoint.

Heat shock is a physiological and environmental stress that leads to the denaturation and inactivation of cellular proteins and is used in hyperthermia cancer therapy. Previously, we revealed that mild heat shock (42 °C) delays the mitotic progression by activating the spindle assembly checkpoint (SAC). However, it is unclear whether SAC activation is maintained at higher temperatures than 42 °C. Here, we demonstrated that a high temperature of 44 °C just before mitotic entry led to a prolonged mitotic delay in the early phase, which was shortened by the SAC inhibitor, AZ3146, indicating SAC activation. Interestingly, mitotic slippage was observed at 44 °C after a prolonged delay but not at 42 °C heat shock. Furthermore, the multinuclear cells were generated by mitotic slippage in 44 °C-treated cells. Immunofluorescence analysis revealed that heat shock at 44 °C reduces the kinetochore localization of MAD2, which is essential for mitotic checkpoint activation, in nocodazole-arrested mitotic cells. These results indicate that 44 °C heat shock causes SAC inactivation even after full activation of SAC and suggest that decreased localization of MAD2 at the kinetochore is involved in heat shock-induced mitotic slippage, resulting in multinucleation. Since mitotic slippage causes drug resistance and chromosomal instability, we propose that there may be a risk of cancer malignancy when the cells are exposed to high temperatures.

Functional differences between Hsp105/110 family proteins in cell proliferation, cell division, and drug sensitivity.

The mammalian HSP105/110 family consists of four members, including Hsp105 and Apg-1, which function as molecular chaperones. Recently, we reported that Hsp105 knockdown increases sensitivity to the DNA-damaging agent Adriamycin but decreases sensitivity to the microtubule-targeting agent paclitaxel. However, whether the other Hsp105/110 family proteins have the same functional property is unknown. Here, we show that Apg-1 has different roles from Hsp105 in cell proliferation, cell division, and drug sensitivity. We generated the Apg-1-knockdown HeLa S3 cells by lentiviral expression of Apg-1-targeting short hairpin RNA. Knockdown of Apg-1 but not Hsp105 decreased cell proliferation. Apg-1 knockdown increased cell death upon Adriamycin treatment without affecting paclitaxel sensitivity. The cell synchronization experiment suggests that Apg-1 functions in mitotic progression at a different mitotic subphase from Hsp105, which cause difference in paclitaxel sensitivity. Since Apg-1 is overexpressed in certain types of tumors, Apg-1 may become a potential therapeutic target for cancer treatment without causing resistance to the microtubule-targeting agents.

Combination Treatment of OSI-906 with Aurora B Inhibitor Reduces Cell Viability via Cyclin B1 Degradation-Induced Mitotic Slippage.

Insulin-like growth factor 1 receptor (IGF1R), a receptor-type tyrosine kinase, transduces signals related to cell proliferation, survival, and differentiation. We recently reported that OSI-906, an IGF1R inhibitor, in combination with the Aurora B inhibitor ZM447439 suppresses cell proliferation. However, the mechanism underlying this suppressive effect is yet to be elucidated. In this study, we examined the effects of combination treatment with OSI-906 and ZM447439 on cell division, so as to understand how cell proliferation was suppressed. Morphological analysis showed that the combination treatment generated enlarged cells with aberrant nuclei, whereas neither OSI-906 nor ZM447439 treatment alone caused this morphological change. Flow cytometry analysis indicated that over-replicated cells were generated by the combination treatment, but not by the lone treatment with either inhibitors. Time-lapse imaging showed mitotic slippage following a severe delay in chromosome alignment and cytokinesis failure with furrow regression. Furthermore, in S-trityl-l-cysteine-treated cells, cyclin B1 was precociously degraded. These results suggest that the combination treatment caused severe defect in the chromosome alignment and spindle assembly checkpoint, which resulted in the generation of over-replicated cells. The generation of over-replicated cells with massive aneuploidy may be the cause of reduction of cell viability and cell death. This study provides new possibilities of cancer chemotherapy.

Involvement of Stat3 phosphorylation in mild heat shock-induced thermotolerance.

Thermotolerance is a phenomenon in which cells become resistant to stress by prior exposure to heat shock, and its development is associated with the induction of heat shock proteins (Hsps), including Hsp70. We previously showed that the expression of Hsp70 is regulated by the cytokine signaling transcription factor Stat3, but the role of Stat3 in thermotolerance is not known. In this study, we examined the possible involvement of Stat3 in the acquisition of thermotolerance. We found that severe heat shock-induced morphological changes and decreases in cell viability, which were suppressed by exposure to non-lethal mild heat shock prior to severe heat shock. This thermotolerance development was accompanied by Stat3 phosphorylation and the induction of Hsps such as Hsp105, Hsp70, and Hsp27. Stat3 phosphorylation and Hsp induction were inhibited by AG490, an inhibitor of JAK tyrosine kinase. Consistent with this, we found that mild heat shock-induced thermotolerance was partially suppressed by AG490 or knockdown of Hsp105. We also found that the Stat3 inhibitor Stattic suppresses the acquisition of thermotolerance by inhibiting the mild heat shock-induced Stat3 phosphorylation and Hsp105 expression. These results suggest that the mild heat shock-dependent stimulation of the JAK-Stat signaling pathway contributes to the development of thermotolerance via the induction of Hsps including Hsp105. This signaling pathway may be a useful target for hyperthermia cancer therapy.

SH2D4A promotes centrosome maturation to support spindle microtubule formation and mitotic progression.

Mitotic progression requires the precise formation of spindle microtubules based on mature centrosomes. During the G2/M transition, centrosome maturation progresses, and associated microtubules bundle to form mitotic spindle fibers and capture the chromosomes for alignment at the cell equator. Mitotic kinases-induced phosphorylation signaling is necessary for these processes. Here, we identified SH2 domain-containing protein 4A (SH2D4A/PPP1R38) as a new mitotic regulator. SH2D4A knockdown delays mitotic progression. The time-lapse imaging analysis showed that SH2D4A specifically contributes to the alignment of chromosomes. The cold treatment assay and microtubule regrowth assay indicated that SH2D4A promotes microtubule nucleation to support kinetochore-microtubule attachment. This may be due to the centrosome maturation by SH2D4A via centrosomal recruitment of pericentriolar material (PCM) such as cep192, γ-tubulin, and PLK1. SH2D4A was found to be a negative regulator of PP1 phosphatase. Consistently, treatment with a PP1 inhibitor rescues SH2D4A-knockdown-induced phenotypes, including the microtubule nucleation and centrosomal recruitment of active PLK1. These results suggest that SH2D4A is involved in PCM recruitment to centrosomes and centrosome maturation through attenuation of PP1 phosphatases, accelerating the spindle formation and supporting mitotic progression.