Epithelial aPKC deficiency leads to stem cell loss preceding metaplasia in colorectal cancer initiation.

The early mechanisms of spontaneous tumor initiation that precede malignancy are largely unknown. We show that reduced aPKC levels correlate with stem cell loss and the induction of revival and metaplastic programs in serrated- and conventional-initiated premalignant lesions, which is perpetuated in colorectal cancers (CRCs). Acute inactivation of PKCλ/ι in vivo and in mouse organoids is sufficient to stimulate JNK in non-transformed intestinal epithelial cells (IECs), which promotes cell death and the rapid loss of the intestinal stem cells (ISCs), including those that are LGR5+. This is followed by the accumulation of revival stem cells (RSCs) at the bottom of the crypt and fetal-metaplastic cells (FMCs) at the top, creating two spatiotemporally distinct cell populations that depend on JNK-induced AP-1 and YAP. These cell lineage changes are maintained during cancer initiation and progression and determine the aggressive phenotype of human CRC, irrespective of their serrated or conventional origin.

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer.

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

Whole-mount staining of mouse colorectal cancer organoids and fibroblast-organoid co-cultures.

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment. For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).1.

Hyaluronan driven by epithelial aPKC deficiency remodels the microenvironment and creates a vulnerability in mesenchymal colorectal cancer.

Mesenchymal colorectal cancer (mCRC) is microsatellite stable (MSS), highly desmoplastic, with CD8+ T cells excluded to the stromal periphery, resistant to immunotherapy, and driven by low levels of the atypical protein kinase Cs (aPKCs) in the intestinal epithelium. We show here that a salient feature of these tumors is the accumulation of hyaluronan (HA) which, along with reduced aPKC levels, predicts poor survival. HA promotes epithelial heterogeneity and the emergence of a tumor fetal metaplastic cell (TFMC) population endowed with invasive cancer features through a network of interactions with activated fibroblasts. TFMCs are sensitive to HA deposition, and their metaplastic markers have prognostic value. We demonstrate that in vivo HA degradation with a clinical dose of hyaluronidase impairs mCRC tumorigenesis and liver metastasis and enables immune checkpoint blockade therapy by promoting the recruitment of B and CD8+ T cells, including a proportion with resident memory features, and by blocking immunosuppression.

LncRNA NDRG1 aggravates osteosarcoma progression and regulates the PI3K/AKT pathway by sponging miR-96-5p.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumors in children and adolescents. Large numbers of studies have focused on the long non-coding RNA (lncRNA) that plays essential roles in the progression of osteosarcoma. Nevertheless, the functions and underlying mechanisms of LncRNA NDRG1 in osteosarcoma remain unknown. METHODS: Differentially expressed lncRNAs between osteosarcoma and adjacent normal tissues were identified through RNA sequencing. The role of LncRNA NDRG1 in osteosarcoma proliferation and metastasis were investigated through in vitro and in vivo functional experiments. The interaction between LncRNA NDRG1 and miR-96-5p was verified through bioinformatic analysis and luciferase reporter assay. Regulation relationship between LncRNA NDRG1 and miR-96-5p was further evaluated by the rescue experiments. Additionally, the changes in the expression of epithelial-mesenchymal transition (EMT) and the PI3K/AKT pathway were verified by Western blot. RESULTS: LncRNA NDRG1 was up-regulated in osteosarcoma cell lines and tissues and the expression of LncRNA NDRG1 was correlated with the overall survival of osteosarcoma patients. Functional experiments exhibited that LncRNA NDRG1 aggravated osteosarcoma proliferation and migration in vitro; meanwhile, animals experiments showed that LncRNA NDRG1 promoted osteosarcoma growth and metastasis in vivo. Mechanistically, LncRNA NDRG1 was found to aggravate osteosarcoma progression and regulate the PI3K/AKT pathway by sponging miR-96-5p. CONCLUSIONS: LncRNA NDRG1 aggravates osteosarcoma progression and regulates the PI3K/AKT pathway by sponging miR-96-5p. Therefore, LncRNA NDRG1 could act as a prognostic marker and a therapeutic target for osteosarcoma in the future.

Effect of Angiogenesis in Bone Tissue Engineering.

The reconstruction of large skeletal defects is still a tricky challenge in orthopedics. The newly formed bone tissue migrates sluggishly from the periphery to the center of the scaffold due to the restrictions of exchange of oxygen and nutrition impotent cells osteogenic differentiation. Angiogenesis plays an important role in bone reconstruction and more and more studies on angiogenesis in bone tissue engineering had been published. Promising advances of angiogenesis in bone tissue engineering by scaffold designs, angiogenic factor delivery, in vivo prevascularization and in vitro prevascularization are discussed in detail. Among all the angiogenesis mode, angiogenic factor delivery is the common methods of angiogenesis in bone tissue engineering and possible research directions in the future.

GTP hydrolysis promotes disassembly of the atlastin crossover dimer during ER fusion.

Membrane fusion of the ER is catalyzed when atlastin GTPases anchored in opposing membranes dimerize and undergo a crossed over conformational rearrangement that draws the bilayers together. Previous studies have suggested that GTP hydrolysis triggers crossover dimerization, thus directly driving fusion. In this study, we make the surprising observations that WT atlastin undergoes crossover dimerization before hydrolyzing GTP and that nucleotide hydrolysis and Pi release coincide more closely with dimer disassembly. These findings suggest that GTP binding, rather than its hydrolysis, triggers crossover dimerization for fusion. In support, a new hydrolysis-deficient atlastin variant undergoes rapid GTP-dependent crossover dimerization and catalyzes fusion at an initial rate similar to WT atlastin. However, the variant cannot sustain fusion activity over time, implying a defect in subunit recycling. We suggest that GTP binding induces an atlastin conformational change that favors crossover dimerization for fusion and that the input of energy from nucleotide hydrolysis promotes complex disassembly for subunit recycling.