Periodic Reporting for period 4 - LIPOMET (Dietary Influences on Metastasis: How, When, and Why)
Reporting period: 2023-02-01 to 2024-03-31
Tumor cells that harbor mutations that confer metastatic potential nonetheless still require non-mutational influences to generate macrometastases. This is important, since it suggests that factors derived from our lifestyle could exert a strong influence on tumor progression, and that such factors could be modulated if understood. Yet, we know very little about the nature of the factors that promote metastasis, their origin, how they promote colonization of distant sites, or why not all tumor cells respond to them similarly. What determines whether a particular tumor eventually generates macroscopic metastatic growth after long periods of latency? As a first step towards addressing these critical questions, we have identified a population of metastasis-initiating cells (MICs) in several types of human tumors and have determined that these are highly sensitive to dietary fatty acids (FAs). This has allowed us to start identifying the intriguing characteristics that distinguish metastatic from non-metastatic cells.
Thus, our results indicate that metastatic cells are very sensitive to their metabolic environment, especially to the content and concentration of dietary fat, unearthing a previously overlooked link between what we eat and metastasis. Moreover, they pose the possibility that our diet exerts a strong long-term influence on the propensity of tumors to metastasize. We find this premise very interesting scientifically, albeit socially worrying for several reasons:
1. A very high amount of added fat is consumed regularly through processed food in established and newly industrialized countries.
2. The percentage of clinically obese and overweight people has nearly doubled in the last 30 years, with more than 600 million adults considered obese, and 41 million children under the age of 5 overweight or obese, in 2014 (http://www.who.int/mediacentre/factsheets/fs311(opens in new window)). The phenomenon is occurring worldwide, especially in Europe and even in countries that have historically favored a Mediterranean diet, underscoring the fact that eating habits have dramatically changed with industrialization (www.CRUK.org).
Based on these facts, we hypothesize that specific FAs present in our diets might favor the development of metastatic tumors by:
(a) Modulating the epigenetic landscape of adult stem cells to favor a long-term pro-metastatic metabolic and signalling state.
(b) Modulating the epigenetic landscape of adult stem cells, thereby potentially altering the mutational topography in their cognate tumors.
Thus, our results indicate that metastatic cells are very sensitive to their metabolic environment, especially to the content and concentration of dietary fat, unearthing a previously overlooked link between what we eat and metastasis. Moreover, they pose the possibility that our diet exerts a strong long-term influence on the propensity of tumors to metastasize. We find this premise very interesting scientifically, albeit socially worrying for several reasons:
1. A very high amount of added fat is consumed regularly through processed food in established and newly industrialized countries.
2. The percentage of clinically obese and overweight people has nearly doubled in the last 30 years, with more than 600 million adults considered obese, and 41 million children under the age of 5 overweight or obese, in 2014 (http://www.who.int/mediacentre/factsheets/fs311(opens in new window)). The phenomenon is occurring worldwide, especially in Europe and even in countries that have historically favored a Mediterranean diet, underscoring the fact that eating habits have dramatically changed with industrialization (www.CRUK.org).
Based on these facts, we hypothesize that specific FAs present in our diets might favor the development of metastatic tumors by:
(a) Modulating the epigenetic landscape of adult stem cells to favor a long-term pro-metastatic metabolic and signalling state.
(b) Modulating the epigenetic landscape of adult stem cells, thereby potentially altering the mutational topography in their cognate tumors.
OBJECTIVE 1: We stimulated human oral squamous cell carcinoma cells with palmitic, oleic or linoleic acid, for 4 days in culture prior to orthotopically inoculating them into mice fed with a standard diet. Of these three major dietary components, only palmitic acid strongly induced the cell surface expression of CD36, as well as the penetrance and size of metastatic lesions, without affecting primary tumor initiation. Conversely, oleic acid did not stimulate CD36, and even significantly inhibited metastasis, whereas linoleic acid was innocuous towards both parameters. Thus, oral cancer metastatic cells are selective sensitive to palmitic acid rather than non-specifically stimulated by any fatty acid.
OBJECTIVE 2: We have obtained the quantitative palmitoylated proteome of oral cancer cell lines stimulated with palmitic acid. We are identifying which proteins are altered by palmitic acid in the cell membrane, cytoplasm, nucleus and chromatin-bound fraction. Intriguingly, we have identified important metabolic enzymes associated to the chromatin. We have obtained an ERC Proof of Concept to further develop this part of the project. We have now identified a palmitoyl-transferase as very important for the metastatic initiation and progression. In the ERC PoC we are conducting a large-scale screening of more than 160000 compounds to identify novel inhibitors of this enzyme that could potentially be used in the future as metastatic therapies against oral cancer, melanoma and breast cancer.
OBJECTIVE 3: We have performed deep single cell RNA-seq of several tumours (tumor cells and the tumor storm), and we have managed to obtain the first spatial single cell transcriptomic map of primary tutors versus their metastasis (oral cancer and melanoma). We have now mapped the 3D interactions of the tumors cells at different regions of the tumors and the different stromal components they interact with (vascular and lymphatic, neural cells/Schwann cells, immune etc). This is providing us with the first comprehensive map of the 3D architecture and transcriptome of metastases and how they are influenced by the fatty acids present in them. We have focussed on changes in a subset of neutrophils, macrophages and Schwann cells that are associates exclusively to the lung metastatic lesions. We are conducting functional assays in our in vivo models to delete some of the pathways as altered in these stromal populations to test their potential involvement in metastatic progression.
OBJECTIVE 4: We have stimulated oral cancer cells with palmitic acid, but instead of directly inoculating them in vivo, we washed out the palmitic acid from the medium and grew the cells under standard culture conditions for 14 days. These cells were much more competent in generating metastases than their controls. The prometastatic effect of palmitic acid was so strong that CD36bright cells retained it when serially transplanted. We next orthotopically inoculated oral cancer cells, and once the primary tumors had appeared, we fed the mice for only ten days with a diet rich in either palm oil or olive oil containing high concentrations of palmitic and oleic acid, respectively. Mice were then switched back to their control diet, and at the endpoint primary tumors were collected and serially transplanted into mice fed with a control, non-fat rich, diet. The primary tumors of these three different dietary conditions contained similar numbers of CD36bright cells. However, the tumor cells exposed to 10 days of palm oil-rich diet in the primary recipients displayed a dramatic increase in metastatic competency in the secondary recipient mice. We performed Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of the histone marks indicative of promoters, enhancers, and Polycomb-mediated repression. Intriguingly, we observed stable changes in H3K4me3 in the promoters of approximately 1000 genes.
Interestingly, we have found that this epigenetic memory pertains to a promotion of tumour innervation and particularly to the activation of tumor-associated Schwann cells. We have now characterised the heterogeneity of these Schwann cells by single cell transcriptomics and have identified the signalling nodes that establish their communication with tumor cells. These Schwann cells adopt a pro-regenerative signature that secretes a specialised type of extracellular matrix (known as perineuronal nets) which we have shown to be essential for the metastatic spreading of metastatic stem cells.
OBJECTIVE 2: We have obtained the quantitative palmitoylated proteome of oral cancer cell lines stimulated with palmitic acid. We are identifying which proteins are altered by palmitic acid in the cell membrane, cytoplasm, nucleus and chromatin-bound fraction. Intriguingly, we have identified important metabolic enzymes associated to the chromatin. We have obtained an ERC Proof of Concept to further develop this part of the project. We have now identified a palmitoyl-transferase as very important for the metastatic initiation and progression. In the ERC PoC we are conducting a large-scale screening of more than 160000 compounds to identify novel inhibitors of this enzyme that could potentially be used in the future as metastatic therapies against oral cancer, melanoma and breast cancer.
OBJECTIVE 3: We have performed deep single cell RNA-seq of several tumours (tumor cells and the tumor storm), and we have managed to obtain the first spatial single cell transcriptomic map of primary tutors versus their metastasis (oral cancer and melanoma). We have now mapped the 3D interactions of the tumors cells at different regions of the tumors and the different stromal components they interact with (vascular and lymphatic, neural cells/Schwann cells, immune etc). This is providing us with the first comprehensive map of the 3D architecture and transcriptome of metastases and how they are influenced by the fatty acids present in them. We have focussed on changes in a subset of neutrophils, macrophages and Schwann cells that are associates exclusively to the lung metastatic lesions. We are conducting functional assays in our in vivo models to delete some of the pathways as altered in these stromal populations to test their potential involvement in metastatic progression.
OBJECTIVE 4: We have stimulated oral cancer cells with palmitic acid, but instead of directly inoculating them in vivo, we washed out the palmitic acid from the medium and grew the cells under standard culture conditions for 14 days. These cells were much more competent in generating metastases than their controls. The prometastatic effect of palmitic acid was so strong that CD36bright cells retained it when serially transplanted. We next orthotopically inoculated oral cancer cells, and once the primary tumors had appeared, we fed the mice for only ten days with a diet rich in either palm oil or olive oil containing high concentrations of palmitic and oleic acid, respectively. Mice were then switched back to their control diet, and at the endpoint primary tumors were collected and serially transplanted into mice fed with a control, non-fat rich, diet. The primary tumors of these three different dietary conditions contained similar numbers of CD36bright cells. However, the tumor cells exposed to 10 days of palm oil-rich diet in the primary recipients displayed a dramatic increase in metastatic competency in the secondary recipient mice. We performed Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of the histone marks indicative of promoters, enhancers, and Polycomb-mediated repression. Intriguingly, we observed stable changes in H3K4me3 in the promoters of approximately 1000 genes.
Interestingly, we have found that this epigenetic memory pertains to a promotion of tumour innervation and particularly to the activation of tumor-associated Schwann cells. We have now characterised the heterogeneity of these Schwann cells by single cell transcriptomics and have identified the signalling nodes that establish their communication with tumor cells. These Schwann cells adopt a pro-regenerative signature that secretes a specialised type of extracellular matrix (known as perineuronal nets) which we have shown to be essential for the metastatic spreading of metastatic stem cells.
- How metastatic cells alter the metabolism of the whole organism for their own advantage and how this leads to cancer-associated cachexia.
- How does the epigenetic machinery understands the entrance of palmitic acid in the metastatic cell? How are they molecularly connected?
- Can we target certain pathways expressed in tumour-associated Schwann cells as novel anti-metastatic therapies?
- How does the epigenetic machinery understands the entrance of palmitic acid in the metastatic cell? How are they molecularly connected?
- Can we target certain pathways expressed in tumour-associated Schwann cells as novel anti-metastatic therapies?