Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma.

The tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain-of-oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient-derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate-harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC-derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum-based therapy.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Hsc70 is required for E-cadherin expression in epithelial-like NRK-52E cells.

Heat-shock cognate protein 70 (Hsc70), a molecular chaperone, is involved in multiple cellular functions. We previously demonstrated that Hsc70 is required for TGF-ß-induced Smad signaling in mesenchymal-like NRK-49F cells. In the present study, to compare the Hsc70 functions in TGF-ß-related signaling between epithelial and mesenchymal cells, we examined the effect of Hsc70 downregulation on TGF-ß-induced signaling in epithelial-like NRK-52E cells. TGF-ß-induced Smad signaling was suppressed in cells treated with small interfering RNA (siRNA) for Hsc70. Interestingly, despite interference with TGF-ß signaling, TGF-ß-induced suppression of E-cadherin expression was not affected by Hsc70 knockdown. Instead, Hsc70 knockdown itself caused the suppression of E-cadherin expression at the transcription level in cells treated with Hsc70 siRNA. We also examined the effects of Hsc70 knockdown on the level of E-cadherin-gene repressors, such as Snail1, Slug, Zeb1, Zeb2, and Twist1, and found that transcription of the repressors was upregulated after 24- or 36-h treatment with Hsc70 siRNA. Collectively, these results indicate that, in addition to a supportive role in TGF-ß-induced signaling, Hsc70 supports E-cadherin expression through downregulation of the E-cadherin-gene repressors in NRK-52E cells, suggesting that Hsc70 plays a functional role to maintain the epithelial cell phenotype.

New Swallowing Evaluation Using Piezoelectricity in Normal Individuals.

This study aimed to elucidate the relationship between the piezoelectric waveform latency, hyoid bone movement, surface electromyogram (sEMG), and the pharyngeal transit time (PTT) during swallowing. Forty-one healthy subjects were divided into three age groups: younger (20-39 years, n = 8), middle-aged (40-59 years, n = 9), and older (60-79 years, n = 24). Motion analysis of the hyoid bone using videofluorography (VF), waveform analysis of the front neck using piezoelectric films, and sEMG of the suprahyoid muscle group were performed simultaneously. Latencies of the three movement phases were defined as upward (VFS1), forward (VFS2), and returning to starting position (VFS3). The three phases of the piezoelectric waveform-from wave initiation of the negative wave to the start of the second deep negative wave; from the start of the second deep negative wave to the start of the last positive wave (SLPW); and from the SLPW to the end of the last positive wave-were defined as PS1, PS2, and PS3, respectively. VFS1-3 and PS1-3 were significantly correlated. VFS1 and PS1 latencies were significantly longer with thick liquid than with thin liquid. VFS2, PS1, and PS2 latencies were longer in the older group than in the other two groups. The start of PS1 was nearly equal to those of sEMG and VFS1. Bolus arrival time in the valleculae was statistically equal to the end of the PS1 with both thin and thick liquids. To establish the swallowing screening using Piezoelectric film, further investigation is necessary in the dysphagia patients.

Detectability of neural tracts and nuclei in the brainstem utilizing 3DAC-PROPELLER.

Despite clinical importance of identifying exact anatomical location of neural tracts and nuclei in the brainstem, no neuroimaging studies have validated the detectability of these structures. The aim of this study was to assess the detectability of the structures using three-dimensional anisotropy contrast-periodically rotated overlapping parallel lines with enhanced reconstruction (3DAC-PROPELLER) imaging. Forty healthy volunteers (21 males, 19 females; 19-53 years, average 23.4 years) participated in this study. 3DAC-PROPELLER axial images were obtained with a 3T-MR system at four levels of the brainstem: the lower midbrain, upper and lower pons, and medulla oblongata. Three experts independently judged whether five tracts (corticospinal tract, medial lemniscus, medial longitudinal fasciculus, central tegmental and spinothalamic tracts) and 10 nuclei (oculomotor and trochlear nuclei, spinal trigeminal, abducens, facial, vestibular, hypoglossal, prepositus, and solitary nuclei, locus ceruleus, superior and inferior olives) on each side could be identified. In total, 240 assessments were made. The five tracts and eight nuclei were identified in all the corresponding assessments, whereas the locus ceruleus and superior olive could not be identified in 3 (1.3%) and 16 (6.7%) assessments, respectively. 3DAC-PROPELLER seems extremely valuable imaging method for mapping out surgical strategies for brainstem lesions.