Targeting lipid metabolism in cancer metastasis.

This review delves into the most recent research on the metabolic adaptability of cancer cells and examines how their metabolic functions can impact their progression into metastatic forms. We emphasize the growing significance of lipid metabolism and dietary lipids within the tumor microenvironment, underscoring their influence on tumor progression. Additionally, we present an outline of the interplay between metabolic processes and the epigenome of cancer cells, underscoring the importance regarding the metastatic process. Lastly, we examine the potential of targeting metabolism as a therapeutic approach in combating cancer progression, shedding light on innovative drugs/targets currently undergoing preclinical evaluation.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

The Rho guanosine nucleotide exchange factors Vav2 and Vav3 modulate epidermal stem cell function.

It is known that Rho GTPases control different aspects of the biology of skin stem cells (SSCs). However, little information is available on the role of their upstream regulators under normal and tumorigenic conditions in this process. To address this issue, we have used here mouse models in which the activity of guanosine nucleotide exchange factors of the Vav subfamily has been manipulated using both gain- and loss-of-function strategies. These experiments indicate that Vav2 and Vav3 regulate the number, functional status, and responsiveness of hair follicle bulge stem cells. This is linked to gene expression programs related to the reinforcement of the identity and the quiescent state of normal SSCs. By contrast, in the case of cancer stem cells, they promote transcriptomal programs associated with the identity, activation state, and cytoskeletal remodeling. These results underscore the role of these Rho exchange factors in the regulation of normal and tumor epidermal stem cells.

Dietary palmitic acid promotes a prometastatic memory via Schwann cells.

Fatty acid uptake and altered metabolism constitute hallmarks of metastasis1,2, yet evidence of the underlying biology, as well as whether all dietary fatty acids are prometastatic, is lacking. Here we show that dietary palmitic acid (PA), but not oleic acid or linoleic acid, promotes metastasis in oral carcinomas and melanoma in mice. Tumours from mice that were fed a short-term palm-oil-rich diet (PA), or tumour cells that were briefly exposed to PA in vitro, remained highly metastatic even after being serially transplanted (without further exposure to high levels of PA). This PA-induced prometastatic memory requires the fatty acid transporter CD36 and is associated with the stable deposition of histone H3 lysine 4 trimethylation by the methyltransferase Set1A (as part of the COMPASS complex (Set1A/COMPASS)). Bulk, single-cell and positional RNA-sequencing analyses indicate that genes with this prometastatic memory predominantly relate to a neural signature that stimulates intratumoural Schwann cells and innervation, two parameters that are strongly correlated with metastasis but are aetiologically poorly understood3,4. Mechanistically, tumour-associated Schwann cells secrete a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation. Both the PA-induced memory of this proneural signature and its long-term boost in metastasis require the transcription factor EGR2 and the glial-cell-stimulating peptide galanin. In summary, we provide evidence that a dietary metabolite induces stable transcriptional and chromatin changes that lead to a long-term stimulation of metastasis, and that this is related to a proregenerative state of tumour-activated Schwann cells.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

VAV2 signaling promotes regenerative proliferation in both cutaneous and head and neck squamous cell carcinoma.

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.

The contributions of cancer cell metabolism to metastasis.

Metastasis remains the leading cause of cancer-related deaths worldwide, and our inability to identify the tumour cells that colonize distant sites hampers the development of effective anti-metastatic therapies. However, with recent research advances we are beginning to distinguish metastasis-initiating cells from their non-metastatic counterparts. Importantly, advances in genome sequencing indicate that the acquisition of metastatic competency does not involve the progressive accumulation of driver mutations; moreover, in the early stages of tumorigenesis, cancer cells harbour combinations of driver mutations that endow them with metastatic competency. Novel findings highlight that cells can disseminate to distant sites early during primary tumour growth, remaining dormant and untreatable for long periods before metastasizing. Thus, metastatic cells must require local and systemic influences to generate metastases. This hypothesis suggests that factors derived from our lifestyle, such as our diet, exert a strong influence on tumour progression, and that such factors could be modulated if understood. Here, we summarize the recent findings on how specific metabolic cues modulate the behaviour of metastatic cells and how they influence the genome and epigenome of metastatic cells. We also discuss how crosstalk between metabolism and the epigenome can be harnessed to develop new anti-metastatic therapies.

Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis.

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression data sets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in OvCa cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Targeting metastasis-initiating cells through the fatty acid receptor CD36.

The fact that the identity of the cells that initiate metastasis in most human cancers is unknown hampers the development of antimetastatic therapies. Here we describe a subpopulation of CD44bright cells in human oral carcinomas that do not overexpress mesenchymal genes, are slow-cycling, express high levels of the fatty acid receptor CD36 and lipid metabolism genes, and are unique in their ability to initiate metastasis. Palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36+ metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis in immunodeficient or immunocompetent orthotopic mouse models of human oral cancer, with no side effects. Clinically, the presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas, and inhibition of CD36 also impairs metastasis, at least in human melanoma- and breast cancer-derived tumours. Together, our results indicate that metastasis-initiating cells particularly rely on dietary lipids to promote metastasis.

A complex secretory program orchestrated by the inflammasome controls paracrine senescence.

Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-ß family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-ß ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.

Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity.

Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.

Nonoverlapping functions of the Polycomb group Cbx family of proteins in embryonic stem cells.

Polycomb group proteins are essential regulators of cell fate decisions during embryogenesis. In mammals, at least five different Cbx proteins (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8) are known to associate with the core Polycomb repressive complex 1 (PRC1). Here we show that pluripotency and differentiation of mouse embryonic stem cells (ESCs) is regulated by different Cbx-associated PRC1 complexes with unique functions. Maintenance of pluripotency primarily depends on Cbx7, while lineage commitment is orchestrated by Cbx2 and Cbx4. At the molecular level, we have uncovered a Polycomb autoregulatory loop in which Cbx7 represses the expression of prodifferentiation Cbx proteins, thereby maintaining the pluripotent state. We additionally show that the occupancy of Cbx7 on promoters is completely dependent on PRC2 activity but only partially dependent on a functional PRC1 complex. Thus, Cbx proteins confer distinct target selectivity to the PRC1 complex, achieving a balance between the self-renewal and the differentiation of ESCs.

The circadian molecular clock creates epidermal stem cell heterogeneity.

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

KAP degradation by calpain is associated with CK2 phosphorylation and provides a novel mechanism for cyclosporine A-induced proximal tubule injury.

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice. Since we reported that KAP protects against CsA toxicity in cultured proximal tubule cells, we hypothesized that low KAP levels found in kidneys of CsA-treated mice might correlate with proximal tubule cell injury. To test this hypothesis, we used KAP Tg mice developed in our laboratory and showed that these mice are more resistant to CsA-induced tubular injury than control littermates. Furthermore, we found that calpain, which was activated by CsA in cell cultures and kidney, is involved in KAP degradation and observed that phosphorylation of serine and threonine residues found in KAP PEST sequences by protein kinase CK2 enhances KAP degradation by calpain. Moreover, we also observed that CK2 inhibition protected against CsA-induced cytotoxicity. These findings point to a novel mechanism for CsA-induced kidney toxicity that might be useful in developing therapeutic strategies aimed at preventing tubular cell damage while maintaining the immunosuppressive effects of CsA.

Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation.

Increased oxidative stress, the renin-angiotensin system, and sympathetic overactivation induce hypertension in kidney androgen-regulated protein transgenic mice.

Gender differences in the incidence and severity of hypertension have suggested the involvement of a sex-dependent mechanism. Transgenic (Tg) mice overexpressing kidney androgen-regulated protein (KAP) specifically in kidney showed hypertension associated with oxidative stress. Reactive oxygen species (ROS) are strongly implicated in the pathological signaling leading to hypertension in a framework that includes renin-angiotensin system (RAS) activation, increased sympathetic activity, and cardiac remodeling. In this report, we observed that plasma levels of angiotensin II and catecholamines were increased in KAP Tg mice, compared with wild-type animals. Systemic administration of Tempol, a membrane-permeative superoxide dismutase mimetic, reduced arterial pressure as well as urinary excretion of oxidative stress markers and reduced both angiotensin II and norepinephrine plasma levels in KAP Tg mice. Intracerebroventricular administration of Tempol also reduced arterial pressure in Tg mice. Moreover, administration of apocynin and DPI, inhibitors of NADPH oxidase, a major source of ROS, also reduced arterial pressure and both angiotensin II and norepinephrine plasma levels in Tg mice. Thus, we analyzed the involvement of the RAS and sympathetic nervous system in KAP Tg mouse hypertension. Both captopril and losartan reduced arterial blood pressure in Tg mice, as also occurred after ß-adrenergic blockade with atenolol. Also, intracerebroventricular losartan administration reduced arterial pressure in KAP Tg mice. Our data demonstrate that hypertension in male KAP Tg mice is based on increased oxidative stress, increased sympathetic activity, and RAS activation. Moreover, our results suggest a role for increased oxidative stress in the CNS as a major cause of hypertension in these animals.

Jarid2 regulates mouse epidermal stem cell activation and differentiation.

Jarid2 is required for the genomic recruitment of the polycomb repressive complex-2 (PRC2) in embryonic stem cells. However, its specific role during late development and adult tissues remains largely uncharacterized. Here, we show that deletion of Jarid2 in mouse epidermis reduces the proliferation and potentiates the differentiation of postnatal epidermal progenitors, without affecting epidermal development. In neonatal epidermis, Jarid2 deficiency reduces H3K27 trimethylation, a chromatin repressive mark, in epidermal differentiation genes previously shown to be targets of the PRC2. However, in adult epidermis Jarid2 depletion does not affect interfollicular epidermal differentiation but results in delayed hair follicle (HF) cycling as a consequence of decreased proliferation of HF stem cells and their progeny. We conclude that Jarid2 is required for the scheduled proliferation of epidermal stem and progenitor cells necessary to maintain epidermal homeostasis.