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Targeting the KRAS pathway is a promising but challenging approach for colorectal cancer therapy. Despite showing potent efficacy in BRAF-mutated melanoma, MEK inhibitors appeared to be tolerated by colorectal cancer cells due to their intrinsic compensatory signaling. Here, we performed genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitor to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. Several genes were identified as potential functional drivers, which were significantly enriched in the GRB7-mediated RTK pathway. Loss-of-function and gain-of-function assays validated that GRB7 potently rendered CRC cells primary resistance to MEK inhibitors through the RTK pathway. Mass spectrum analysis of GRB7 immunoprecipitates revealed that PLK1 was the predominant interacting kinase of GRB7. Inhibition of PLK1 suppressed downstream signaling of RTK, including FAK, STAT3, AKT, and 4EBP1. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. In conclusion, we identified GRB7-PLK1 as a pivotal axis mediating RTKs, resulting in MEK inhibitor tolerance. PLK1 is therefore a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients harboring KRAS mutations.

Cancer immunotherapy using immune-checkpoint blockade has displayed promising clinical effects, but prevalent antibody-based inhibitors face multiple challenges such as low response rate, acquired resistance, and adverse effects. The intracellular expression of PD-1/PD-L1 in recycling endosomes and their active trafficking to membrane highlight the importance of depleting rather than interfering with checkpoint proteins. Preclinical investigations on the therapeutic effects of lead compounds that function by degrading immune checkpoint ligands and receptors have reported highly promising results. By harnessing the degradation capabilities of the lysosome, proteasome and autophagosomes, different small molecules and peptides potently induced degradation of checkpoint proteins and enhanced anti-tumor immunity. Both in vitro and in vivo experiments support the therapeutic efficacy of these molecules. Thus, targeted degradation through endo-lysosomal, autophagic, proteasomal, or endoplasmic reticulum-related pathways may provide promising strategies for tackling the challenges in cancer immunotherapy.

Metastatic melanoma is the most aggressive and obstinate skin cancer with poor prognosis. Variant novel applicable regimens have emerged during the past decades intensively, while the most profound approaches are oncogene-targeted therapy and T-lymphocyte mediated immunotherapy. Although targeted therapies generated remarkable and rapid clinical responses in the majority of patients, acquired resistance was developed promptly within months leading to tumor relapse. By contrast, immunotherapies elicited long-term tumor regression. However, the overall response rate was limited. In view of the above, either targeted therapy or immunotherapy cannot elicit durable clinical responses in large range of patients. Interestingly, the advantages and limitations of these regimens happened to be complementary. An increasing number of preclinical studies and clinical trials proved a synergistic antitumor effect with the combination of targeted therapy and immunotherapy, implying a promising prospect for the treatment of metastatic melanoma. In order to achieve a better therapeutic effectiveness and reduce toxicity in patients, great efforts need to be made to illuminate multifaceted interplay between targeted therapy and immunotherapy.

Checkpoint blockade therapy targeting the programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 promotes T-cell-mediated immunosurveillance against tumours, and has been associated with marked clinical benefit in cancer patients. Antibodies against PD-L1 function by blocking PD-L1 on the cell surface, but intracellular storage of PD-L1 and its active redistribution to the cell membrane can minimize the therapeutic benefits, which highlights the importance of targeting PD-L1 throughout the whole cell. Here, we show that PD-L1 is palmitoylated in its cytoplasmic domain, and that this lipid modification stabilizes PD-L1 by blocking its ubiquitination, consequently suppressing PD-L1 degradation by lysosomes. We identified palmitoyltransferase ZDHHC3 (DHHC3) as the main acyltransferase required for the palmitoylation of PD-L1, and show that the inhibition of PD-L1 palmitoylation via 2-bromopalmitate, or the silencing of DHHC3, activates antitumour immunity in vitro and in mice bearing MC38 tumour cells. We also designed a competitive inhibitor of PD-L1 palmitoylation that decreases PD-L1 expression in tumour cells to enhance T-cell immunity against the tumours. These findings suggest new strategies for overcoming PD-L1-mediated immune evasion in cancer. The physiological degradation of programmed-death ligand 1 is reduced by the palmitoylation of its intracellular domain, and this process can be inhibited to promote T-cell immunity against tumours.

Heat shock protein 90-α (Hsp90α) is an intracellular molecular chaperone. However, it can also be secreted with the underlying regulatory mechanism remaining far from clear. Here we show that the secreted Hsp90α is a C-terminal truncated form and its secretion is regulated by the C-terminal EEVD motif via interacting with proteins containing tetratricopeptide repeat domains. We also demonstrate that secretion of Hsp90α is determined by the phosphorylation status at residue Thr-90, regulated by protein kinase A and protein phosphatase 5. We further demonstrate that the secretion of Hsp90α is a prerequisite for its proinvasiveness function and blocking the secreted Hsp90α results in significant inhibition of tumor metastasis. Meanwhile, the level of plasma Hsp90α is positively correlated with tumor malignancy in clinical cancer patients. In sum, our results reveal the regulatory mechanism of Hsp90α secretion, and its function in tumor invasiveness, indicating it can be a promising diagnostic marker for tumor malignancy in clinical application.

The adenoma-carcinoma sequence is a well-accepted roadmap for the development of sporadic colorectal cancer. However, cellular heterogeneity in aberrant epithelial cells limits our understanding of carcinogenesis in colorectal tissues. Here, we performed a single-cell RNA sequencing survey of 54,788 cells from patient-matched tissue samples, including blood, normal tissue, para-cancer, polyp, and colorectal cancer. At each stage of carcinogenesis, we characterized cell types, transcriptional signatures, and differentially expressed genes of distinct cell populations. The molecular signatures of epithelial cells at normal, benign, and malignant stages were defined at the single-cell scale. Adenoma and carcinoma precursor cell populations were identified and characterized followed by validation with large cohort biopsies. Protein tyrosine kinases (PTKs) BMX and HCK were identified as potential drivers of adenoma initiation. Specific BMX and HCK upregulations were observed in adenoma precursor cell populations from normal and adenoma biopsies. Overexpression of BMX and HCK significantly promoted colorectal epithelial cell proliferation. Importantly, in the organoid culture system, BMX and HCK upregulations resulted in the formation of multilayered polyp-like buds protruding towards the organoid lumen, mimicking the pathological polyp morphology often observed in colorectal cancer. Molecular mechanism analysis revealed that upregulation of BMX or HCK activated the JAK-STAT pathway. In conclusion, our work improved the current knowledge regarding colorectal epithelial evolution during carcinogenesis at the single-cell resolution. These findings may lead to improvements in colorectal cancer diagnosis and treatment.

A sensitive and specific diagnosis biomarker, in principle scalable to most cancer types, is needed to reduce the prevalent cancer mortality. Meanwhile, the investigation of diagnosis determinants of a biomarker will facilitate the interpretation of its screening results in clinic. Here we design a large‐scale (1558 enrollments), multicenter (multiple hospitals), and cross‐validation (two datasets) clinic study to validate plasma Hsp90α quantified by ELISA as a pan‐cancer biomarker. ROC curve shows the optimum diagnostic cutoff is 69.19 ng/mL in discriminating various cancer patients from all controls (AUC 0.895, sensitivity 81.33% and specificity 81.65% in test cohort; AUC 0.893, sensitivity 81.72% and specificity 81.03% in validation cohort). Similar results are noted in detecting early‐stage cancer patients. Plasma Hsp90α maintains also broad‐spectrum for cancer subtypes, especially with 91.78% sensitivity and 91.96% specificity in patients with AFP‐limited liver cancer. In addition, we demonstrate levels of plasma Hsp90α are determined by ADAM10 expression, which will affect Hsp90α content in exosomes. Furthermore, Western blotting and PRM‐based quantitative proteomics identify that partial false ELISA‐negative patients secret high levels of plasma Hsp90α. Mechanism analysis reveal that TGFβ‐PKCγ gene signature defines a distinct pool of hyperphosphorylated Hsp90α at Theronine residue. In clinic, a mechanistically relevant population of false ELISA‐negative patients express also higher levels of PKCγ. In sum, plasma Hsp90α is a novel pan‐cancer diagnosis biomarker, and cancer diagnosis with plasma Hsp90α is particularly effective in those patients with high expression of ADAM10, but may be insufficient to detect the patients with low ADAM10 and those with hyperphosphorylated Hsp90α. Early diagnosis of cancer generally increases the chances for successful treatment by focusing on detecting symptomatic patients as early as possible. Our study has proved that plasma Hsp90α is a novel pan‐cancer diagnosis biomarker, even for cancer patients at early‐stage. The extension of plasma Hsp90α in clinic will reduce the mortality caused by the prevalent cancer disease. In addition, the examination that the molecular features associated with the secretion and detection of extracellular Hsp90α provides fundamental implications to interpreting the screening results and improving the diagnosis performance for all the biomarkers used in clinic.

Although clinical trials have shown that RAF inhibitors prolong the survival of patients with BRAF-mutant melanoma, resistance inevitably develops; resistance is shown here to be frequently mediated by the expression of splicing variants of mutant BRAF. Mechanism of RAF inhibitor resistance Although recent clinical trials have shown the efficacy of B-RAF inhibitors in the treatment of melanomas with activating B-RAF mutations, the patients inevitably develop resistance. David Solit and colleagues now identify a mechanism of acquired resistance conferred by a structural change in B-RAF itself. The expression of a 61-kilodalton splice variant of mutant B-RAF leads to enhanced B-RAF dimerization, rendering it resistant to kinase inhibitors. This variant was found to be expressed in 6 of 19 patients who had developed resistance to the B-RAF inhibitor PLX4032. Activated RAS promotes dimerization of members of the RAF kinase family 1 , 2 , 3 . ATP-competitive RAF inhibitors activate ERK signalling 4 , 5 , 6 , 7 by transactivating RAF dimers 4 . In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E) 8 . However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4–8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS–GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Breast cancer is a major threat to women’s health worldwide. Although the 5-year survival rate is relatively high, treating recurrent and metastatic breast cancer remains challenging. Existing anticancer drugs are often accompanied by adverse reactions; thus, there is an urgent need to explore safer and more effective treatment options. Marine natural compounds, especially alkaloids, are considered to be a potential treasure trove of new anticancer drugs due to their unique chemical structure and wide range of biological activities. A variety of marine alkaloids against breast cancer, including ecteinascidins, halichondrins, manzamines, and trabectedins, have opened new avenues for breast cancer treatment by employing multiple mechanisms, such as inducing cell apoptosis and autophagy, blocking cell cycle, inhibiting angiogenesis, targeting oncogene pathways, and inhibiting metastasis and invasion. Currently, Yondelis (trabectedin) has completed phase II clinical trials in patients with breast cancer and has shown certain efficacy. However, the clinical application of marine alkaloids still needs further research and development. This article deeply explores the mechanism of action of marine alkaloids against breast cancer and anticipates their clinical application prospects. With the deepening of research and the advancement of development, marine alkaloids are expected to bring new breakthroughs in breast cancer treatment.

The subcortical maternal complex (SCMC) plays a crucial role in early embryonic development. Malfunction of SCMC leads to reproductive diseases in women. However, the molecular function and assembly basis for SCMC remain elusive. Here we reconstituted mouse SCMC and solved the structure at atomic resolution using single-particle cryo-electron microscopy. The core complex of SCMC was formed by MATER, TLE6 and FLOPED, and MATER embraced TLE6 and FLOPED via its NACHT and LRR domains. Two core complexes further dimerize through interactions between two LRR domains of MATERs in vitro. FILIA integrates into SCMC by interacting with the carboxyl-terminal region of FLOPED. Zygotes from mice with Floped C-terminus truncation showed delayed development and resembled the phenotype of zygotes from Filia knockout mice. More importantly, the assembly of mouse SCMC was affected by corresponding clinical variants associated with female reproductive diseases and corresponded with a prediction based on the mouse SCMC structure. Our study paves the way for further investigations on SCMC functions during mammalian preimplantation embryonic development and reveals underlying causes of female reproductive diseases related to SCMC mutations, providing a new strategy for the diagnosis of female reproductive disorders. Here, using cryo-electron microscopy and functional assays in mouse oocytes and embryos, the authors delineate the composition of the subcortical maternal complex, showing that clinical variants associated with female reproductive diseases disrupt complex formation.