Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-β receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

Obesity is a leading risk factor for cancer. However, understanding the crosstalk between adipocytes and tumor cells in vivo, independently of dietary contributions, is a major gap in the field. Here we used a prostate cancer (PCa) mouse model in which the signaling adaptor p62/Sqstm1 is selectively inactivated in adipocytes. p62 loss in adipocytes results in increased osteopontin secretion, which mediates tumor fatty acid oxidation and invasion, leading to aggressive metastatic PCa in vivo. Furthermore, p62 deficiency triggers in adipocytes a general shutdown of energy-utilizing pathways through mTORC1 inhibition, which supports nutrient availability for cancer cells. This reveals a central role of adipocyte's p62 in the symbiotic adipose tissue-tumor collaboration that enables cancer metabolic fitness.

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).

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).

Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during reactive oxygen species (ROS) generation, and how ROS promote tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) λ/ι loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC through cell-autonomous and non-autonomous mechanisms. Although PKCλ/ι promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKCλ/ι levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.

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).

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

Activation of non-shivering thermogenesis is considered a promising approach to lower body weight in obesity. p62 deficiency in adipocytes reduces systemic energy expenditure but its role in sustaining mitochondrial function and thermogenesis remains unresolved. NBR1 shares a remarkable structural similarity with p62 and can interact with p62 through their respective PB1 domains. However, the physiological relevance of NBR1 in metabolism, as compared to that of p62, was not clear. Here we show that whole-body and adipocyte-specific ablation of NBR1 reverts the obesity phenotype induced by p62 deficiency by restoring global energy expenditure and thermogenesis in brown adipose tissue. Impaired adrenergic-induced browning of p62-deficient adipocytes is rescued by NBR1 inactivation, unveiling a negative role of NBR1 in thermogenesis under conditions of p62 loss. We demonstrate that upon p62 inactivation, NBR1 represses the activity of PPARγ, establishing an unexplored p62/NBR1-mediated paradigm in adipocyte thermogenesis that is critical for the control of obesity.

Intestinal epithelial homeostasis requires continuous renewal supported by stem cells located in the base of the crypt. Disruption of this balance results in failure to regenerate and initiates tumorigenesis. The β-catenin and Yap pathways in Lgr5+ stem cells have been shown to be central to this process. However, the precise mechanisms by which these signaling molecules are regulated in the stem cell population are not totally understood. Protein kinase C ζ (PKCζ) has been previously demonstrated to be a negative regulator of intestinal tumorigenesis. Here, we show that PKCζ suppresses intestinal stem cell function by promoting the downregulation of β-catenin and Yap through direct phosphorylation. PKCζ deficiency results in increased stem cell activity in organoid cultures and in vivo, accounting for the increased tumorigenic and regenerative activity response of Lgr5+-specific PKCζ-deficient mice. This demonstrates that PKCζ is central to the control of stem cells in intestinal cancer and homeostasis.

Cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. We show that the upregulation of SOX2 is central to this process, which is repressed by protein kinase Cζ (PKCζ). PKCζ deficiency activates the reprogramming of colonic fibroblasts to generate a predominant SOX2-dependent CAF population expressing the WNT regulator Sfrp2 as its top biomarker. SOX2 directly binds the Sfrp1/2 promoters, and the inactivation of Sox2 or Sfrp1/2 in CAFs impaired the induction of migration and invasion of colon cancer cells, as well as their tumorigenicity in vivo. Importantly, recurrence-free and overall survival of colorectal cancer (CRC) patients negatively correlates with stromal PKCζ levels. Also, SOX2 expression in the stroma is associated with CRC T invasion and worse prognosis of recurrence-free survival. Therefore, the PKCζ-SOX2 axis emerges as a critical step in the control of CAF pro-tumorigenic potential.

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.

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.

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.

Hepatomegaly can be triggered by insulin and insulin-unrelated etiologies. Insulin acts via AKT, but how other challenges cause hepatomegaly is unknown.

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.

The activation of the transcription factor NF-kappaB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IkappaB, NF-kappaB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In zeta protein kinase C (PKC)-deficient cells, NF-kappaB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-alpha) is severely impaired. In vitro assays showed that zetaPKC directly phosphorylates RelA. Here we demonstrate that Ser311 accounts for zetaPKC phosphorylation of RelA and that this site is phosphorylated in vivo in response to TNF-alpha. Also, an inactivating mutation of that residue severely impairs RelA transcriptional activity, blocks its anti-apoptotic function and abrogates the interaction of RelA with the co-activator CBP as well as its recruitment, and that of RNA polymerase II (Pol II) with the interleukin-6 (IL-6) promoter. The interaction of endogenous CBP with endogenous RelA is inhibited in zetaPKC-/- cells, as well as the binding of Pol II to the IL-6 promoter. These results demonstrate the mechanism whereby zetaPKC regulates NF-kappaB activation in vivo.

Tumors undergo nutrient stress and need to reprogram their metabolism to survive. The stroma may play a critical role in this process by providing nutrients to support the epithelial compartment of the tumor. Here we show that p62 deficiency in stromal fibroblasts promotes resistance to glutamine deprivation by the direct control of ATF4 stability through its p62-mediated polyubiquitination. ATF4 upregulation by p62 deficiency in the stroma activates glucose carbon flux through a pyruvate carboxylase-asparagine synthase cascade that results in asparagine generation as a source of nitrogen for stroma and tumor epithelial proliferation. Thus, p62 directly targets nuclear transcription factors to control metabolic reprogramming in the microenvironment and repress tumorigenesis, and identifies ATF4 as a synthetic vulnerability in p62-deficient tumor stroma.

Increasingly effective therapies targeting the androgen receptor have paradoxically promoted the incidence of neuroendocrine prostate cancer (NEPC), the most lethal subtype of castration-resistant prostate cancer (PCa), for which there is no effective therapy. Here we report that protein kinase C (PKC)λ/ι is downregulated in de novo and during therapy-induced NEPC, which results in the upregulation of serine biosynthesis through an mTORC1/ATF4-driven pathway. This metabolic reprogramming supports cell proliferation and increases intracellular S-adenosyl methionine (SAM) levels to feed epigenetic changes that favor the development of NEPC characteristics. Altogether, we have uncovered a metabolic vulnerability triggered by PKCλ/ι deficiency in NEPC, which offers potentially actionable targets to prevent therapy resistance in PCa.

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.