Establishment of patient-derived organoids for guiding personalized therapies in breast cancer patients.

Breast cancer has become the most commonly diagnosed cancer. The intra- and interpatient heterogeneity induced a considerable variation in treatment efficacy. There is an urgent requirement for preclinical models to anticipate the effectiveness of individualized drug responses. Patient-derived organoids (PDOs) can accurately recapitulate the architecture and biological characteristics of the origin tumor, making them a promising model that can overtake many limitations of cell lines and PDXs. However, it is still unclear whether PDOs-based drug testing can benefit breast cancer patients, particularly those with tumor recurrence or treatment resistance. Fresh tumor samples were surgically resected for organoid culture. Primary tumor samples and PDOs were subsequently subjected to H&E staining, immunohistochemical (IHC) analysis, and whole-exome sequencing (WES) to make comparisons. Drug sensitivity tests were performed to evaluate the feasibility of this model for predicting patient drug response in clinical practice. We established 75 patient-derived breast cancer organoid models. The results of H&E staining, IHC, and WES revealed that PDOs inherited the histologic and genetic characteristics of their parental tumor tissues. The PDOs successfully predicted the patient's drug response, and most cases exhibited consistency between PDOs' drug susceptibility test results and the clinical response of the matched patient. We conclude that the breast cancer organoids platform can be a potential preclinical tool used for the selection of effective drugs and guided personalized therapies for patients with advanced breast cancer.

The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

Crenobacter oryzisoli sp. nov., a novel phosphate-solubilizing bacterium isolated from the paddy soil.

Two Gram-staining-negative, facultative anaerobic, rod-shaped and phosphate-solubilizing strains designated SG2303T and SG2305, were isolated from paddy soil in China. Phylogenetic analysis based on 16 S rRNA gene sequences indicated that SG2303T and SG2305 represented a member of the genus Crenobacter within the family Neisseriaceae of the phylum Pseudomonadota. Strain SG2303T displayed higher 16 S rRNA gene sequence similarities with members of the genus Crenobacter ranging from 93.5 to 94.0%. Strains C. luteus YIM 78141T and C. cavernae K1W11S-77T were closest related to the isolated strains and were considered as type strains. Growth of strain SG2303T occurred at 10-55 °C (optimum 37 °C), pH 5.0-9.0 (optimum pH 6.0-7.0) and 0-1% (w/v) NaCl (optimum 0%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG2303T and its closely related taxa were 76.1-78.2% and 20.5-22.1%, respectively. The genomic DNA G + C content was 62.2%. The quinone of strain SG2303T was Q-8. The major fatty acids (> 10%) of strain SG2303T were C16:0 (30.6%), summed feature 3 (C16:1ω7c and/or C16:1ω6c) (26.0%) and C12:0 3OH (12.1%). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phospholipids (PL), glycolipid (GL) and unidentified lipids (UL). Based on the results of the phylogenetic, physiological, biochemical, and morphological analysis, strain SG2303T is recognized as a novel species of the genus Crenobacter, for which the name Crenobacter oryzisoli sp. nov. is proposed. The type strain is SG2303T (= GDMCC 1.3970T = JCM 36468T). In addition, SG2303T was also able of phosphorus solubilization and promoting the growth of rice seeds. Strain SG2303T exhibited a relatively high dissolvable phosphorus content of 2.52 µg·mL- 1.

Pan-Cancer Analysis of the Prognostic and Immunological Role of TOMM40 to Identify Its Function in Breast Cancer.

Breast cancer (BRCA) is currently the most commonly diagnosed malignancy in women worldwide. Previous studies have demonstrated that mitophagy is important for the prevention and treatment of BRCA. However, few studies have focused on the individual mitochondrial autophagy-related genes (MARG) in human cancers. Based on bioinformatics analyses, TOMM40 was identified as a prognostic DEMARG (PDEMARGs); Kaplan-Meier (KM) survival analysis also indicates that TOMM40 can be useful as a prognostic indicator in BRCAs, with patients in the high expression group having a poorer prognosis. For 20 distinct cancer kinds, there were appreciable differences in the expression of TOMM40 between tumor and normal tissues; in addition, in 21 different cancer types, there were associations between the expression profile of TOMM40 and patient prognosis. Gene Set Enrichment Analysis (GSEA), functional enrichment analysis, and immunological and drug sensitivity analyses of TOMM40 have indicated its biological significance in pan-cancers. Knockdown of TOMM40 in MDA-MB-231 cells inhibited their proliferation, migration, and invasiveness. In conclusion, we found that TOMM40 has prognostic value in 21 cancers, including breast cancer, by bioinformatics analysis. Based on immune correlation analysis, TOMM40 may also be a potential immunotherapeutic target for the treatment of BRCA. Therefore, our results may provide researchers to further explore the role of MARGs, especially TOMM40, in the developmental process of breast cancer, which may provide new directions and targets for the improvement of prognosis of breast cancer patients and their treatment.

Sporophytic control of tapetal development and pollen fertility by a mitogen-activated protein kinase cascade in rice.

Tapetum, the innermost layer of the anther wall, provides essential nutrients and materials for pollen development. Timely degradation of anther tapetal cells is a prerequisite for normal pollen development in flowering plants. Tapetal cells facilitate male gametogenesis by providing cellular contents after highly coordinated programmed cell death (PCD). Tapetal development is regulated by a transcriptional network. However, the signaling pathway(s) involved in this process are poorly understood. In this study, we report that a mitogen-activated protein kinase (MAPK) cascade composed of OsYDA1/OsYDA2-OsMKK4-OsMPK6 plays an important role in tapetal development and male gametophyte fertility. Loss of function of this MAPK cascade leads to anther indehiscence, enlarged tapetum, and aborted pollen grains. Tapetal cells in osmkk4 and osmpk6 mutants exhibit an increased presence of lipid body-like structures within the cytoplasm, which is accompanied by a delayed occurrence of PCD. Expression of a constitutively active version of OsMPK6 (CA-OsMPK6) can rescue the pollen defects in osmkk4 mutants, confirming that OsMPK6 functions downstream of OsMKK4 in this pathway. Genetic crosses also demonstrated that the MAPK cascade sporophyticly regulates pollen development. Our study reveals a novel function of rice MAPK cascade in plant male reproductive biology.

Baicalein enhances immune response in TNBC by inhibiting leptin expression of adipocytes.

Triple-negative breast cancer (TNBC) is a special pathological type of breast cancer (BC) with poor prognosis. Obesity is shown to be involved in TNBC tumor progression. The interaction between obesity and BC has generated great attention in recent years, however, the mechanism is still unclear. Here, we showed that leptin secreted by adipocytes upregulated PD-L1 expression in TNBC through the p-STAT3 signaling pathway and that baicalein inhibited PD-L1 expression in tumor microenvironment by suppressing leptin transcription of adipocytes. Collectively, our findings suggest that leptin may be the key factor participating in obesity-related tumor progression and that baicalein can break through the dilemma to boost the anti-tumor immune response.

Novel models by machine learning to predict prognosis of breast cancer brain metastases.

BACKGROUND: Breast cancer brain metastases (BCBM) are the most fatal, with limited survival in all breast cancer distant metastases. These patients are deemed to be incurable. Thus, survival time is their foremost concern. However, there is a lack of accurate prediction models in the clinic. What's more, primary surgery for BCBM patients is still controversial. METHODS: The data used for analysis in this study was obtained from the SEER database (2010-2019). We made a COX regression analysis to identify prognostic factors of BCBM patients. Through cross-validation, we constructed XGBoost models to predict survival in patients with BCBM. Meanwhile, a BCBM cohort from our hospital was used to validate our models. We also investigated the prognosis of patients treated with surgery or not, using propensity score matching and K-M survival analysis. Our results were further validated by subgroup COX analysis in patients with different molecular subtypes. RESULTS: The XGBoost models we created had high precision and correctness, and they were the most accurate models to predict the survival of BCBM patients (6-month AUC = 0.824, 1-year AUC = 0.813, 2-year AUC = 0.800 and 3-year survival AUC = 0.803). Moreover, the models still exhibited good performance in an externally independent dataset (6-month: AUC = 0.820; 1-year: AUC = 0.732; 2-year: AUC = 0.795; 3-year: AUC = 0.936). Then we used Shiny-Web tool to make our models be easily used from website. Interestingly, we found that the BCBM patients with an annual income of over USD$70,000 had better BCSS (HR = 0.523, 95%CI 0.273-0.999, P < 0.05) than those with less than USD$40,000. The results showed that in all distant metastasis sites, only lung metastasis was an independent poor prognostic factor for patients with BCBM (OS: HR = 1.606, 95%CI 1.157-2.230, P < 0.01; BCSS: HR = 1.698, 95%CI 1.219-2.365, P < 0.01), while bone, liver, distant lymph nodes and other metastases were not. We also found that surgical treatment significantly improved both OS and BCSS in BCBM patients with the HER2 + molecular subtypes and was beneficial to OS of the HR-/HER2- subtype. In contrast, surgery could not help BCBM patients with HR + /HER2- subtype improve their prognosis (OS: HR = 0.887, 95%CI 0.608-1.293, P = 0.510; BCSS: HR = 0.909, 95%CI 0.604-1.368, P = 0.630). CONCLUSION: We analyzed the clinical features of BCBM patients and constructed 4 machine-learning prognostic models to predict their survival. Our validation results indicate that these models should be highly reproducible in patients with BCBM. We also identified potential prognostic factors for BCBM patients and suggested that primary surgery might improve the survival of BCBM patients with HER2 + and triple-negative subtypes.

Causal effects of COVID-19 on cancer risk: A Mendelian randomization study.

In contemporary literature, little attention has been paid to the association between coronavirus disease-2019 (COVID-19) and cancer risk. We performed the Mendelian randomization (MR) to investigate the causal associations between the three types of COVID-19 exposures (critically ill COVID-19, hospitalized COVID-19, and respiratory syndrome coronavirus 2 (SARS-CoV-2) infection) and 33 different types of cancers of the European population. The results of the inverse-variance-weighted model indicated that genetic liabilities to critically ill COVID-19 had suggestive causal associations with the increased risk for HER2-positive breast cancer (odds ratio [OR] = 1.0924; p-value = 0.0116), esophageal cancer (OR = 1.0004; p-value = 0.0226), colorectal cancer (OR = 1.0010; p-value = 0.0242), stomach cancer (OR = 1.2394; p-value = 0.0331), and colon cancer (OR = 1.0006; p-value = 0.0453). The genetic liabilities to hospitalized COVID-19 had suggestive causal associations with the increased risk for HER2-positive breast cancer (OR = 1.1096; p-value = 0.0458), esophageal cancer (OR = 1.0005; p-value = 0.0440) as well as stomach cancer (OR = 1.3043; p-value = 0.0476). The genetic liabilities to SARS-CoV-2 infection had suggestive causal associations with the increased risk for stomach cancer (OR = 2.8563; p-value = 0.0019) but with the decreasing risk for head and neck cancer (OR = 0.9986, p-value = 0.0426). The causal associations of the above combinations were robust through the test of heterogeneity and pleiotropy. Together, our study indicated that COVID-19 had causal effects on cancer risk.

WRKY15 Suppresses Tracheary Element Differentiation Upstream of VND7 During Xylem Formation.

Formation of the vascular cylinder, a structure critical to water and nutrient transport in higher plants, is highly regulated. Here we identify WRKY15 as an important regulator that suppresses tracheary element (TE) differentiation in Arabidopsis (Arabidopsis thaliana). Overexpression of WRKY15 resulted in discontinuous protoxylem vessel files and TEs with reduced spiral wall thickening/lignification. Expression of a dominant-negative WRKY15 variant, WRKY15-EAR, led to extra protoxylem vessels and ectopic TEs with increased spiral wall thickening/lignification. Ectopic TE formation in the root cortex and hypocotyl/leaf epidermis reveals that the suppression of WRKY15 is sufficient to trigger the transdifferentiation of other types of cells to TEs. Expression profiling, RT-qPCR, and reporter analyses revealed that WRKY15 suppresses the expression of VASCULAR-RELATED NAC DOMAIN7 (VND7), a master transcriptional regulator that promotes TE differentiation. We propose that WRKY15 negatively regulates VND7 expression indirectly based on (1) the absence of a W-box in the promoter of VND7 and (2) the observation that WRKY15 and VND7 are expressed in different cells in the vascular cylinder, with WRKY15 expressed in the procambial cells and VND7 in the protoxylem poles of procambium and differentiating TEs. Future research is needed to reveal the details underlying the interaction of WRKY15 and VND7 in plant vascular development.

Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Correction: lncRNA PSMB8-AS1 promotes colorectal cancer progression through sponging miR-1299 to upregulate ADAMTS5.

This corrects the article DOI: 10.4149/neo_2022_220111N42.

Upregulated IncRNA WTAPP1 in Triple Negative Breast Cancer Predicts Survival.

The migration and angiogenesis of endothelial progenitor cells require the involvement of WTAPP1. Cell migration and angiogenesis are critical for cancer development, we therefore speculated that WTAPP1 may participate in cancer biology. This study aimed to explore the role of WTAPP1 in triple-negative breast cancer (TNBC). A total of 68 patients (females, 38 to 67 years old, mean age 52.1 ± 5.9 years old) were enrolled in this study. The effects of over-expression of WTAPP1 and miR-34a on EEF2K were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. Transient cell transfections were performed to explore the interactions between genes. Cell proliferation assay was used to analyze cell proliferation. Transwell assays were performed to detect cell invasive and migration. Flow cytometry was used to evaluate apoptosis. WTAPP1 was upregulated in tumor tissues of TNBC patients and high expression levels of WTAPP1 in tumor tissues predicted poor survival. In contrast, miR-34a was downregulated in TNBC. Correlation analysis showed that WTAPP1 and miR-34a were negatively correlated with each other. In cancer cells, overexpression of WTAPP1 resulted in downregulation of miR-34a. Remarkably, overexpression of WTAPP1 increased the expression levels of EEF2K, a target of miR-34a. Overexpression of WTAPP1 and EEF2K increased proliferation, invasion and migration of TNBC cells, while overexpression of miR-34a showed different results. In addition, overexpression of miR-34a enhanced the effects of overexpression of WTAPP1 and EEF2K on apoptosis. WTAPP1 may promote TNBC cell proliferation by downregulating miR-34a.

Neoadjuvant pyrotinib, trastuzumab, and docetaxel for HER2-positive breast cancer (PHEDRA): a double-blind, randomized phase 3 trial.

BACKGROUND: Pyrotinib (an irreversible pan-ErbB inhibitor) plus capecitabine has survival benefits and acceptable tolerability in patients with HER2-positive metastatic breast cancer. We further assessed addition of pyrotinib to trastuzumab and docetaxel in the neoadjuvant setting. METHODS: In this multicenter, double-blind, phase 3 study (PHEDRA), treatment-naive women with HER2-positive early or locally advanced breast cancer were randomly assigned (1:1) to receive four neoadjuvant cycles of oral pyrotinib or placebo (400 mg) once daily, plus intravenous trastuzumab (8 mg/kg loading dose, followed by 6 mg/kg) and docetaxel (100 mg/m2) every 3 weeks. The primary endpoint was the total pathological complete response (tpCR; ypT0/is and ypN0) rate per independent central review. RESULTS: Between Jul 23, 2018, and Jan 8, 2021, 355 patients were randomly assigned, 178 to the pyrotinib group and 177 to the placebo group. The majority of patients completed four cycles of neoadjuvant treatment as planned (92.7% and 97.7% in the pyrotinib and placebo groups, respectively). The tpCR rate was 41.0% (95% CI 34.0 to 48.4) in the pyrotinib group compared with 22.0% (95% CI 16.6 to 28.7) in the placebo group (difference, 19.0% [95% CI 9.5 to 28.4]; one-sided P < 0.0001). The objective response rate per investigator was 91.6% (95% CI 86.6 to 94.8) in the pyrotinib group and 81.9% (95% CI 75.6 to 86.9) in the placebo group after the neoadjuvant treatment, resulting in an increase of 9.7% (95% CI 2.7 to 16.6). The most common grade 3 or worse adverse events were diarrhea (79 [44.4%] in the pyrotinib group and nine [5.1%] in the placebo group), neutropenia (33 [18.5%] and 36 [20.3%]), and decreased white blood cell count (29 [16.3%] and 24 [13.6%]). No deaths were reported during neoadjuvant treatment. CONCLUSIONS: The primary endpoint of the study was met. Neoadjuvant pyrotinib, trastuzumab, and docetaxel significantly improved the tpCR rate compared with placebo, trastuzumab, and docetaxel, with manageable toxicity, providing a new option for HER2-positive early or locally advanced breast cancer. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03588091.

Expression of a plastid-localized sugar transporter in the suspensor is critical to embryogenesis.

Plant growth and development rely on sugar transport between source and sink cells and between different organelles. The plastid-localized sugar transporter GLUCOSE-6-PHOSPHATE TRANSLOCATER1 (GPT1) is an essential gene in Arabidopsis (Arabidopsis thaliana). Using a partially rescued gpt1 mutant and cell-specific RNAi suppression of GPT1, we demonstrated that GPT1 is essential to the function of the embryo suspensor and the development of the embryo. GPT1 showed a dynamic expression/accumulation pattern during embryogenesis. Inhibition of GPT1 accumulation via RNAi using a suspensor-specific promoter resulted in embryos and seedlings with defects similar to auxin mutants. Loss of function of GPT1 in the suspensor also led to abnormal/ectopic cell division in the lower part of the suspensor, which gave rise to an ectopic embryo, resulting in twin embryos in some seeds. Furthermore, loss of function of GPT1 resulted in vacuolar localization of PIN-FORMED1 (PIN1) and altered DR5 auxin activity. Proper localization of PIN1 on the plasma membrane is essential to polar auxin transport and distribution, a key determinant of pattern formation during embryogenesis. Our findings suggest that the function of GPT1 in the embryo suspensor is linked to sugar and/or hormone distribution between the embryo proper and the maternal tissues, and is important for maintenance of suspensor identity and function during embryogenesis.

The Arabidopsis Pleiotropic Drug Resistance Transporters PEN3 and PDR12 Mediate Camalexin Secretion for Resistance to Botrytis cinerea.

Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

lncRNA PSMB8-AS1 promotes colorectal cancer progression through sponging miR-1299 to upregulate ADAMTS5.

Long non-coding RNAs (lncRNAs) have been reported to be vital participants in tumor progression. Recently, lncRNA PSMB8-AS1 has been uncovered to facilitate pancreatic cancer progression by regulating miR-382-3p/STAT1/PD-L1 network. Nonetheless, the role of PSMB8-AS1 and its underlying mechanism have not been well-explored in colorectal cancer (CRC). The expression of RNAs or proteins was detected via qRT-PCR or western blot assays. Functional assays were involved in evaluating the effects of PSMB8-AS1/miR-1299/ADAMTS5 on the malignant behaviors of CRC cells. The molecular mechanism of PSMB8-AS1 was explored via mechanism analyses in CRC cells. Based on experimental results, PSMB8-AS1 expression was notably higher in CRC cell lines than in normal cells. The downregulation of PSMB8-AS1 repressed cell viability, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC while promoting cell apoptosis. It was also revealed that PSMB8-AS1 could sponge miR-1299 to upregulate ADAMTS5 in CRC cells. In rescue assays, we further discovered that miR-1299 inhibition or ADAMTS5 overexpression abrogated the suppressive influence of PSMB8-AS1 deficiency on CRC cell growth. In addition, PSMB8-AS1 was validated to induce M2 polarization. In conclusion, PSMB8-AS1 sponges miR-1299 to increase PSMB8-AS1 expression, thus promoting CRC cell growth.

Mitogen-activated protein kinase cascades in plant signaling.

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns (DAMPs) or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns (P/MAMPs), pathogen-derived effectors, and environmental factors. In this review, we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity, response to environmental stresses, and normal growth and development. Each tier of the MAPK cascades is encoded by a small gene family, and multiple members can function redundantly in an MAPK cascade. Yet, they carry out a diverse array of biological functions in plants. How the signaling specificity is achieved has become an interesting topic of MAPK research. Future investigations into the molecular mechanism(s) underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus, the spatiotemporal expression patterns of all the components in the signaling pathway, and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development.

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4 (MKK4) and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA (YDA), also known as MAPK KINASE KINASE4 (MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade (YDA-MKK4/MKK5-MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5-MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 pathogen-associated molecular pattern (PAMP) treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9) approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5-MPK3/MPK6 module in both plant immunity and growth/development.

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops.

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.