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.

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.

Air-liquid organotypic assays to investigate cellular crosstalk in the tumor microenvironment of cancer cells.

Air-liquid organotypic culture models enable the study of the cellular crosstalk in the tumor microenvironment. This 3D assay recapitulates the tumor niche more faithfully than 2D culture systems and represents a versatile platform that can be easily adapted to different types of cancer cells, stromal components, or ECM composition. Here, we detail the steps to build an organotypic culture including the preparation of the organotypic structure, organotypic gels, cell seeding, gel casting, membrane processing, and image and data analysis. For complete details on the use and execution of this protocol, please refer to Linares et al. (2022).

The lactate-NAD+ axis activates cancer-associated fibroblasts by downregulating p62.

Reduced p62 levels are associated with the induction of the cancer-associated fibroblast (CAF) phenotype, which promotes tumorigenesis in vitro and in vivo through inflammation and metabolic reprogramming. However, how p62 is downregulated in the stroma fibroblasts by tumor cells to drive CAF activation is an unresolved central issue in the field. Here we show that tumor-secreted lactate downregulates p62 transcriptionally through a mechanism involving reduction of the NAD+/NADH ratio, which impairs poly(ADP-ribose)-polymerase 1 (PARP-1) activity. PARP-1 inhibition blocks the poly(ADP-ribosyl)ation of the AP-1 transcription factors, c-FOS and c-JUN, which is an obligate step for p62 downregulation. Importantly, restoring p62 levels in CAFs by NAD+ renders CAFs less active. PARP inhibitors, such as olaparib, mimick lactate in the reduction of stromal p62 levels, as well as the subsequent stromal activation both in vitro and in vivo, which suggests that therapies using olaparib would benefit from strategies aimed at inhibiting CAF activity.

Obestatin signalling counteracts glucocorticoid-induced skeletal muscle atrophy via NEDD4/KLF15 axis.

BACKGROUND: A therapeutic approach for the treatment of glucocorticoid-induced skeletal muscle atrophy should be based on the knowledge of the molecular mechanisms determining the unbalance between anabolic and catabolic processes and how to re-establish this balance. Here, we investigated whether the obestatin/GPR39 system, an autocrine signalling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against chronic glucocorticoid-induced muscle atrophy. METHODS: In this study, we used an in vivo model of muscle atrophy induced by the synthetic glucocorticoid dexamethasone to examine the liaison molecules that define the interaction between the glucocorticoid receptor and the obestatin/GPR39 systems. The findings were extended to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: KLF15 and FoxO transcription factors were identified as direct targets of obestatin signalling in the control of proteostasis in skeletal muscle. The KLF15-triggered gene expression program, including atrogenes and FoxOs, was regulated via KLF15 ubiquitination by the E3 ubiquitin ligase NEDD4. Additionally, a specific pattern of FoxO post-translational modification, including FoxO4 phosphorylation by Akt pathway, was critical in the regulation of the ubiquitin-proteasome system. The functional cooperativity between Akt and NEDD4 in the regulation of FoxO and KLF15 provides integrated cues to counteract muscle proteostasis and re-establish protein synthesis. CONCLUSIONS: The effective control of FoxO activity in response to glucocorticoid is critical to counteract muscle-related pathologies. These results highlight the potential of the obestatin/GPR39 system to fine-tune the effects of glucocorticoids on skeletal muscle wasting.

Mouse model of colorectal cancer: orthotopic co-implantation of tumor and stroma cells in cecum and rectum.

In vivo interrogation of the functional role of genes implicated in colorectal cancer (CRC) is limited by the need for physiological models that mimic the disease. Here, we describe a protocol that provides the steps required for the orthotopic co-implantation of tumoral and stromal cells into the cecum and rectum to investigate the crosstalk between the tumor and its microenvironment. This protocol recapitulates metastases to the lymph nodes, liver, and lungs observed in human CRC. For complete details on the use and execution of this protocol, please refer to Kasashima et al. (2020).

An Orthotopic Implantation Mouse Model of Hepatocellular Carcinoma with Underlying Liver Steatosis.

This protocol provides the steps required for a mouse liver orthotopic implantation model. The reliable pre-clinical animal models that have similar characteristics to hepatocellular carcinoma (HCC) are a powerful tool to unveil the mechanisms controlling tumor initiation and progression. Here, we describe a syngeneic orthotopic HCC model that recapitulates the role of a host pro-tumorigenic microenvironment by pre-conditioning mouse livers with a high-fat diet (HFD). For complete details on the use and execution of this protocol, please refer to Kudo et al. (2020).

Publisher Correction: Obestatin controls skeletal muscle fiber-type determination.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Improvement of Duchenne muscular dystrophy phenotype following obestatin treatment.

BACKGROUND: This study was performed to test the therapeutic potential of obestatin, an autocrine anabolic factor regulating skeletal muscle repair, to ameliorate the Duchenne muscular dystrophy (DMD) phenotype. METHODS AND RESULTS: Using a multidisciplinary approach, we characterized the ageing-related preproghrelin/GPR39 expression patterns in tibialis anterior (TA) muscles of 4-, 8-, and 18-week-old mdx mice (n = 3/group) and established the effects of obestatin administration at this level in 8-week-old mdx mice (n = 5/group). The findings were extended to in vitro effects on human immortalized DMD myotubes. An analysis of TAs revealed an age-related loss of preproghrelin expression, as precursor of obestatin, in mdx mice. Administration of obestatin resulted in a significant increase in tetanic specific force (33.0% ± 1.5%, P < 0.05), compared with control mdx mice. Obestatin-treated TAs were characterized by reduction of fibres with centrally located nuclei (10.0% ± 1.2%, P < 0.05) together with an increase in the number of type I fibres (25.2% ± 1.7%, P < 0.05) associated to histone deacetylases/myocyte enhancer factor-2 and peroxisome proliferator-activated receptor-gamma coactivator 1α axis, and down-regulation of ubiquitin E3-ligases by inactivation of FoxO1/4, indexes of muscle atrophy. Obestatin reduced the level of contractile damage and tissue fibrosis. These observations correlated with decline in serum creatine kinase (58.8 ± 15.2, P < 0.05). Obestatin led to stabilization of the sarcolemma by up-regulation of utrophin, α-syntrophin, ß-dystroglycan, and α7ß1-integrin proteins. These pathways were also operative in human DMD myotubes. CONCLUSIONS: These results highlight the potential of obestatin as a peptide therapeutic for preserving muscle integrity in DMD, thus allowing a better efficiency of gene or cell therapy in a combined therapeutic approach.

Obestatin controls the ubiquitin-proteasome and autophagy-lysosome systems in glucocorticoid-induced muscle cell atrophy.

BACKGROUND: Many pathological states characterized by muscle atrophy are associated with an increase in circulating glucocorticoids and poor patient prognosis, making it an important target for treatment. The development of treatments for glucocorticoid-induced and wasting disorder-related skeletal muscle atrophy should be designed based on how the particular transcriptional program is orchestrated and how the balance of muscle protein synthesis and degradation is deregulated. Here, we investigated whether the obestatin/GPR39 system, an autocrine/paracrine signaling system acting on myogenesis and with anabolic effects on the skeletal muscle, could protect against glucocorticoid-induced muscle cell atrophy. METHODS: In the present study, we have utilized mouse C2C12 myotube cultures to examine whether the obestatin/GPR39 signaling pathways can affect the atrophy induced by the synthetic glucocorticoid dexamethasone. We have extended these findings to in vitro effects on human atrophy using human KM155C25 myotubes. RESULTS: The activation of the obestatin/GPR39 system protects from glucocorticoid-induced atrophy by regulation of Akt, PKD/PKCµ, CAMKII and AMPK signaling and its downstream targets in the control of protein synthesis, ubiquitin-proteasome system and autophagy-lysosome system in mouse cells. We compared mouse and human myotube cells in their response to glucocorticoid and identified differences in both the triggering of the atrophic program and the response to obestatin stimulation. Notably, we demonstrate that specific patterns of post-translational modifications of FoxO4 and FoxO1 play a key role in directing FoxO activity in response to obestatin in human myotubes. CONCLUSIONS: Our findings emphasize the function of the obestatin/GPR39 system in coordinating a variety of pathways involved in the regulation of protein degradation during catabolic conditions.

Obestatin controls skeletal muscle fiber-type determination.

Obestatin/GPR39 signaling stimulates skeletal muscle growth and repair by inducing both G-protein-dependent and -independent mechanisms linking the activated GPR39 receptor with distinct sets of accessory and effector proteins. In this work, we describe a new level of activity where obestatin signaling plays a role in the formation, contractile properties and metabolic profile of skeletal muscle through determination of oxidative fiber type. Our data indicate that obestatin regulates Mef2 activity and PGC-1α expression. Both mechanisms result in a shift in muscle metabolism and function. The increase in Mef2 and PGC-1α signaling activates oxidative capacity, whereas Akt/mTOR signaling positively regulates myofiber growth. Taken together, these data indicate that the obestatin signaling acts on muscle fiber-type program in skeletal muscle.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.