Identification of an anaplastic subtype of prostate cancer amenable to therapies targeting SP1 or translation elongation.

Histopathological heterogeneity is a hallmark of prostate cancer (PCa). Using spatial and parallel single-nucleus transcriptomics, we report an androgen receptor (AR)-positive but neuroendocrine-null primary PCa subtype with morphologic and molecular characteristics of small cell carcinoma. Such small cell-like PCa (SCLPC) is clinically aggressive with low AR, but high stemness and proliferation, activity. Molecular characterization prioritizes protein translation, represented by up-regulation of many ribosomal protein genes, and SP1, a transcriptional factor that drives SCLPC phenotype and overexpresses in castration-resistant PCa (CRPC), as two potential therapeutic targets in AR-indifferent CRPC. An SP1-specific inhibitor, plicamycin, effectively suppresses CRPC growth in vivo. Homoharringtonine, a Food And Drug Administration-approved translation elongation inhibitor, impedes CRPC progression in preclinical models and patients with CRPC. We construct an SCLPC-specific signature capable of stratifying patients for drug selectivity. Our studies reveal the existence of SCLPC in admixed PCa pathology, which may mediate tumor relapse, and establish SP1 and translation elongation as actionable therapeutic targets for CRPC.

Integrins regulate stemness in solid tumor: an emerging therapeutic target.

Integrins are the adhesion molecules and transmembrane receptors that consist of α and ß subunits. After binding to extracellular matrix components, integrins trigger intracellular signaling and regulate a wide spectrum of cellular functions, including cell survival, proliferation, differentiation and migration. Since the pattern of integrins expression is a key determinant of cell behavior in response to microenvironmental cues, deregulation of integrins caused by various mechanisms has been causally linked to cancer development and progression in several solid tumor types. In this review, we discuss the integrin signalosome with a highlight of a few key pro-oncogenic pathways elicited by integrins, and uncover the mutational and transcriptomic landscape of integrin-encoding genes across human cancers. In addition, we focus on the integrin-mediated control of cancer stem cell and tumor stemness in general, such as tumor initiation, epithelial plasticity, organotropic metastasis and drug resistance. With insights into how integrins contribute to the stem-like functions, we now gain better understanding of the integrin signalosome, which will greatly assist novel therapeutic development and more precise clinical decisions.

Enhanced Expression of Thaumatin-like Protein Gene (LeTLP1) Endows Resistance to Trichoderma atroviride in Lentinula edodes.

Lentinula edodes (shiitake mushrooms) is heavily affected by the infection of Trichoderma atroviride, causing yield loss and decreases quality in shiitake mushrooms. The selection and breeding of fungal-resistant L. edodes species are an important approach to protecting L. edodes from T. atroviride infection. Herein, a highly resistant L. edodes strain (Y3334) and a susceptible strain (Y55) were obtained by using a resistance evaluation test. Transcriptome analyses and qRT-PCR detection showed that the expression level of LeTLP1 (LE01Gene05009) was strongly induced in response to T. atroviride infection in the resistant Y3334. Then, LeTLP1-silenced and LeTLP1-overexpression transformants were obtained. Overexpression of LeTLP1 resulted in resistance to T. atroviride. Compared with the parent strain Y3334, LeTLP1-silenced transformants had reduced resistance relative to T. atroviride. Additionally, the LeTLP1 protein (Y3334) exhibited significant antifungal activity against T. atroviride. These findings suggest that overexpression of LeTLP1 is a major mechanism for the resistance of L. edodes to T. atroviride. The molecular basis provides a theoretical basis for the breeding of resistant L. edodes strains and can eventually contribute to the mushroom cultivation industry and human health.

Effects of Medium Composition and Genetic Background on Agrobacterium-Mediated Transformation Efficiency of Lentinula edodes.

The establishment of genetic transformation method is crucial for the functional genomics research in filamentous fungi. Although the transformation method has been developed in several types of fungi, a highly efficient and convenient transformation system is desperately needed in Lentinula edodes. Present work established the Agrobacterium-mediated transformation (ATMT) of basidiomycete L. edodes in both monokaryon and dikaryon mycelia by using constructed binary plasmid pCAMBIA-1300-GFP. Then, the transformation efficiency of ATMT was evaluated by using different mediums for recipient incubation and different varieties of L. edodes. The results showed that in dikaryon strain W1, the positive hygromycin-resistant transformants was observed in all medium with the positive frequency of selected transformants that ranged from 0 to 30%. While in the monokaryon strain W1-26, only the millet medium group obtained positive transformants with a positive frequency of 75.48%. Moreover, three dikaryotic wild strains (YS55, YS3334, and YS3357) and two dikaryotic cultivated strains (W1 and S606) showed the highest transformation efficiency, with 32.96% of the germination frequency, and 85.12% of positive frequency for hygromycin-resistant transformants. This work demonstrated that Agrobacterium-mediated transformation was successfully performed in L. edodes, and the genotype of recipients as well as the medium for mycelial incubation were suggested to play key roles in determining the transformation efficiency. These findings may provide new avenues for the genetic modification of edible mushroom and may extend the cognition of DNA-mediated transformation in filamentous fungi.

Expression Profile of Laccase Gene Family in White-Rot Basidiomycete Lentinula edodes under Different Environmental Stresses.

Laccases belong to ligninolytic enzymes and play important roles in various biological processes of filamentous fungi, including fruiting-body formation and lignin degradation. The process of fruiting-body development in Lentinula edodes is complex and is greatly affected by environmental conditions. In this paper, 14 multicopper oxidase-encoding (laccase) genes were analyzed in the draft genome sequence of L. edodes strain W1-26, followed by a search of multiple stress-related Cis-elements in the promoter region of these laccase genes, and then a transcription profile analysis of 14 laccase genes (Lelcc) under the conditions of different carbon sources, temperatures, and photoperiods. All laccase genes were significantly regulated by varying carbon source materials. The expression of only two laccase genes (Lelcc5 and Lelcc6) was induced by sodium-lignosulphonate and the expression of most laccase genes was specifically upregulated in glucose medium. Under different temperature conditions, the expression levels of most laccase genes decreased at 39 °C and transcription was significantly increased for Lelcc1, Lelcc4, Lelcc5, Lelcc9, Lelcc12, Lelcc13, and Lelcc14 after induction for 24 h at 10 °C, indicating their involvement in primordium differentiation. Tyrosinase, which is involved in melanin synthesis, was clustered with the same group as Lelcc4 and Lelcc7 in all the different photoperiod treatments. Meanwhile, five laccase genes (Lelcc8, Lelcc9, Lelcc12, Lelcc13, and Lelcc14) showed similar expression profiles to that of two blue light receptor genes (LephrA and LephrB) in the 12 h light/12 h dark treatment, suggesting the involvement of laccase genes in the adaptation process of L. edodes to the changing environment and fruiting-body formation. This study contributes to our understanding of the function of the different Lelcc genes and facilitates the screening of key genes from the laccase gene family for further functional research.

The heat shock protein 40 LeDnaJ regulates stress resistance and indole-3-acetic acid biosynthesis in Lentinula edodes.

DnaJ proteins, termed heat shock proteins based on their molecular weight, function as molecular chaperones that play critical roles in regulating organism growth and development as well as adaptation to the environment. However, little has been reported on their gene function in higher basidiomycetes. Here, the heat shock protein 40 (LeDnaJ) gene was cloned and characterized from Lentinula edodes. RNA interference was used to explore the function of LeDnaJ in response to heat stress and Trichoderma atroviride. Integration of the target gene into the L. edodes genome was confirmed by Southern blot analysis, and the silence efficiency of LeDnaJ was analyzed by qRT-PCR. The results revealed that LeDnaJ silence caused defects in mycelial growth and resistance to heat stress and T. atroviride, but increased the mycelial density compared with the wild type (WT) strain S606. Additionally, the IAA content showed a more than 10-fold increase in the WT after heat stress, but an about two-fold increase in the two LeDnaJ RNAi transfortants (LeDnaJ-i-6 and LeDnaJ-i-8). Previous study has shown that enhanced IAA (indole-3-acetic acid) content enhanced the thermotolerance of the heat-sensitive strain YS3357. In this study, it was documented that IAA amendments could partly restore the resistance to T. atroviride and thermotolerance of the two LeDnaJ RNAi transformants. Overall, LeDnaJ is nvolved in fungal growth, T. atroviride resistance, and thermotolerance by regulating the IAA biosynthesis in L. edodes.

Comparative secretomic analysis of lignocellulose degradation by Lentinula edodes grown on microcrystalline cellulose, lignosulfonate and glucose.

Lentinula edodes has the potential to degrade woody and nonwoody lignocellulosic biomass. However, the mechanism of lignocellulose degradation by L. edodes is unclear. The aim of this work is to explore the profiling of soluble secreted proteins involved in lignocellulose degradation in L. edodes. For that, we compared the secretomes of L. edodes grown on microcrystalline cellulose, cellulose with lignosulfonate and glucose. Based on nanoliquid chromatography coupled with tandem mass spectrometry of whole-protein hydrolysate, 230 proteins were identified. Label-free proteomic analysis showed that the most abundant carbohydrate-active enzymes involved in polysaccharide hydrolysis were endo-ß-1,4-glucanase, α-galactosidase, polygalacturonase and glucoamylase in both cellulosic secretomes. In contrast, enzymes involved in lignin degradation were most abundant in glucose culture, with laccase 1 being the predominant protein (13.13%). When the cellulose and cellulose with lignosulfonate secretomes were compared, the abundance of cellulases and hemicellulases was higher in cellulose with lignosulfonate cultures, which was confirmed by enzyme activity assays. In addition, qRT-PCR analysis demonstrated that the expression levels of genes encoding cellulases and hemicellulases were significantly increased (by 32.2- to 1166.7-fold) when L. edodes was grown in cellulose with lignosulfonate medium. BIOLOGICAL SIGNIFICANCE: In this article, the secretomes of L. edodes grown on three different carbon sources were compared. The presented results revealed the profiling of extracellular enzymes involved in lignocellulose degradation, which is helpful to further explore the mechanism of biomass bioconversion by L. edodes.

Diversity and effect of Trichoderma spp. associated with green mold disease on Lentinula edodes in China.

Lentinula edodes, one of the most important edible mushrooms in China, is affected heavily by the infection of green mold that overgrows mushroom mycelia. We collected the diseased samples from main L. edodes cultivation regions in China to characterize the pathogen and to study the effect of Trichoderma spp. on L. edodes species. We identified six Trichoderma species, that is, T. harzianum, T. atroviride, T. viride, T. pleuroticola, T. longibrachiatum, and T. oblongisporum based on the internal transcribed spacer or tef1-α sequences and morphology characteristics. In confrontation cultures on Petri plates or in tubes, and in L. edodes cultures in a medium containing Trichoderma metabolites, L. edodes mycelia were not only distorted and swollen, but also inhibited by Trichoderma isolates. It is not possible that adjusting pH value or temperature is used for controlling L. edodes green disease, because the growth of most of Trichoderma isolates and L. edodes shared similar pH and temperature conditions.