Metabolic adaptation towards glycolysis supports resistance to neoadjuvant chemotherapy in early triple negative breast cancers.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is the standard of care for patients with early-stage triple negative breast cancers (TNBC). However, more than half of TNBC patients do not achieve a pathological complete response (pCR) after NAC, and residual cancer burden (RCB) is associated with dismal long-term prognosis. Understanding the mechanisms underlying differential treatment outcomes is therefore critical to limit RCB and improve NAC efficiency. METHODS: Human TNBC cell lines and patient-derived organoids were used in combination with real-time metabolic assays to evaluate the effect of NAC (paclitaxel and epirubicin) on tumor cell metabolism, in particular glycolysis. Diagnostic biopsies (pre-NAC) from patients with early TNBC were analyzed by bulk RNA-sequencing to evaluate the predictive value of a glycolysis-related gene signature. RESULTS: Paclitaxel induced a consistent metabolic switch to glycolysis, correlated with a reduced mitochondrial oxidative metabolism, in TNBC cells. In pre-NAC diagnostic biopsies from TNBC patients, glycolysis was found to be upregulated in non-responders. Furthermore, glycolysis inhibition greatly improved response to NAC in TNBC organoid models. CONCLUSIONS: Our study pinpoints a metabolic adaptation to glycolysis as a mechanism driving resistance to NAC in TNBC. Our data pave the way for the use of glycolysis-related genes as predictive biomarkers for NAC response, as well as the development of inhibitors to overcome this glycolysis-driven resistance to NAC in human TNBC patients.

A non-dividing cell population with high pyruvate dehydrogenase kinase activity regulates metabolic heterogeneity and tumorigenesis in the intestine.

Although reprogramming of cellular metabolism is a hallmark of cancer, little is known about how metabolic reprogramming contributes to early stages of transformation. Here, we show that the histone deacetylase SIRT6 regulates tumor initiation during intestinal cancer by controlling glucose metabolism. Loss of SIRT6 results in an increase in the number of intestinal stem cells (ISCs), which translates into enhanced tumor initiating potential in APCmin mice. By tracking down the connection between glucose metabolism and tumor initiation, we find a metabolic compartmentalization within the intestinal epithelium and adenomas, where a rare population of cells exhibit features of Warburg-like metabolism characterized by high pyruvate dehydrogenase kinase (PDK) activity. Our results show that these cells are quiescent cells expressing +4 ISCs and enteroendocrine markers. Active glycolysis in these cells suppresses ROS accumulation and enhances their stem cell and tumorigenic potential. Our studies reveal that aerobic glycolysis represents a heterogeneous feature of cancer, and indicate that this metabolic adaptation can occur in non-dividing cells, suggesting a role for the Warburg effect beyond biomass production in tumors.

Microenvironment-driven intratumoral heterogeneity in head and neck cancers: clinical challenges and opportunities for precision medicine.

Squamous cell carcinoma of the head and neck (SCCHN) is among the most prevalent cancer types worldwide. Despite multimodal therapeutic approaches that include surgical resection, radiation therapy or concurrent chemoradiation, targeted therapy and immunotherapy, SCCHN is still associated with a poor prognosis for patients with locally advanced or recurrent/metastatic (R/M) diseases. Although next-generation sequencing data from thousands of SCCHN patients have provided a comprehensive landscape of the somatic genomic alterations in this disease, genomic-based precision medicine is not implemented yet in routine clinical use since no satisfactory genetic biomarker has been identified for diagnosis, patient outcome prediction and selection of tailored therapeutic options. The lack of significant improvement in SCCHN patient survival over the last decades stresses the need for reliable predictive biomarkers and new therapeutic strategies for personalized clinical management of SCCHN patients. Targeting the SCCHN-associated microenvironment or the interaction of the latter with cancer cells may represent such paradigm shift in the development of new strategies to treat SCCHN patients, as exemplified by the recent implementation of immune checkpoint inhibitors to improve clinical outcomes by increasing anti-tumor immune responses in SCCHN patients. Several clinical trials are in progress in SCCHN patients to evaluate the activity of monoclonal antibodies and small-molecule inhibitors targeting the tumor microenvironment (TME) at different treatment settings, including combinations with adjuvant surgery, radiation therapy and chemotherapy. This review describes the current knowledge about the influence of the TME on intratumoral heterogeneity and clinical relapse in human SCCHN patients. More precisely, the role of hypoxia as well as the presence of non-cancer cells (e.g. cancer-associated fibroblasts and immune cells) on therapy response of SCCHN cells is highlighted. We also discuss relevant (pre)clinical models that may help integrate the microenvironment-tumor cell interplay in translational research studies for SCCHN. Finally, this review explores potential therapeutic strategies that may exploit the crosstalk between TME and SCCHN cells in order to implement fundamental changes in the tumor treatment paradigm of patients with locally advanced or R/M SCCHN.

Eicosapentaenoic acid prevents salt sensitivity in diabetic rats and decreases oxidative stress.

OBJECTIVES: Salt sensitivity (SS) is associated with increased cardiovascular risk in patients with Type 2 diabetes mellitus (T2-DM) due to an increase in renal oxidation. ω-3 polyunsaturated fatty acids have shown antioxidant effects, but a typical Western diet contains limited content. In particular, ω-3 polyunsaturated fatty acids are able to activate nuclear factor erythroid 2-related factor 2 (Nrf-2) to prevent diabetes mellitus-related complications by mitigating oxidative stress. Therefore, we hypothesized that eicosapentaenoic acid (EPA; ω-3) modulates SS in rats with T2-DM by decreasing renal oxidative stress via Nrf-2 activation and enhancing the antiinflammatory response via interleukin (IL) 6 modulation. METHODS: Three-month-old male rats (n = 40) were fed with a Normal Na-diet (NNaD) and randomly selected into four groups: Healthy Wistar nondiabetic rats (Wi), diabetic controls (eSS), arachidonic acid-treated eSS (AA; ω-6), and EPA-treated eSS (ω-3). After 1 year, rats were placed in metabolic cages for 7 d and fed a NNaD, followed by a 7-d period with a High Na-diet (HNaD). Systolic blood pressure, body weight, serum IL-6 and reactive oxygen species (ROS) levels were determined at the end of each 7-d period. Glycated hemoglobin (HbA1c), triacylglycerol, creatinine, and cholesterol levels were determined. ROS levels and Nrf-2 expression in kidney lysates were also assayed. Histologic changes were evaluated. A t test or analysis of variance was used for the statistical analysis. RESULTS: After a HNaD, systolic blood pressure increased in both the control eSS and AA groups, but not in the EPA and Wi groups. However, HbA1c levels remained unchanged by the treatments, which suggests that the observed beneficial effect was independent of HbA1c levels. The IL-6 levels were higher in the eSS and AA groups, but remained unaltered in EPA and Wi rats after a HNaD diet. Interestingly, EPA protected against serum ROS in rats fed the HNaD, whereas AA did not. In kidney lysates, ROS decreased significantly in the EPA group compared with the eSS group, and Nrf-2 expression was consistently higher compared with the AA and eSS groups. Diabetic rats presented focal segmental sclerosis, adherence to Bowman capsule, and mild-to-moderate interstitial fibrosis. EPA and AA treatment prevented kidney damage. CONCLUSIONS: An adequate ω3-to-ω6 ratio prevents SS in diabetic rats by a mechanism that is independent of glucose metabolism but associated with the prevention of renal oxidative stress generation. These data suggest that EPA antioxidant properties may prevent the development of hypertension or kidney damage.

Activation of RSK by phosphomimetic substitution in the activation loop is prevented by structural constraints.

The activation of the majority of AGC kinases is regulated by two phosphorylation events on two conserved serine/threonine residues located on the activation loop and on the hydrophobic motif, respectively. In AGC kinase family, phosphomimetic substitutions with aspartate or glutamate, leading to constitutive activation, have frequently occurred at the hydrophobic motif site. On the contrary, phosphomimetic substitutions in the activation loop are absent across the evolution of AGC kinases. This observation is explained by the failure of aspartate and glutamate to mimic phosphorylatable serine/threonine in this regulatory site. By detailed 3D structural simulations of RSK2 and further biochemical evaluation in cells, we show that the phosphomimetic residue on the activation loop fails to form a critical salt bridge with R114, necessary to reorient the αC-helix and to activate the protein. By a phylogenetic analysis, we point at a possible coevolution of a phosphorylatable activation loop and the presence of a conserved positively charged amino acid on the αC-helix. In sum, our analysis leads to the unfeasibility of phosphomimetic substitution in the activation loop of RSK and, at the same time, highlights the peculiar structural role of activation loop phosphorylation.

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis.

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.

Increased dietary levels of α-linoleic acid inhibit mammary tumor growth and metastasis.

OBJECTIVE: The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma. MATERIALS AND METHODS: BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma. RESULTS: After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERß in tumor lysates. CONCLUSION: These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.

Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid.

Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells. We demonstrated that down-regulation of the transcription factor glioma-associated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AA-induced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches.

A potential role of PUFAs and COXIBs in cancer chemoprevention.

Polyunsaturated fatty acids (PUFAs), particularly the ω-3 PUFAs and COXIBs have been associated with decreased inflammation and the prevention of tumorigenesis. ω-3 PUFAs have shown to display multiple antitumour actions, while ω-6 PUFAs and its derived eicosanoids promote the effects in cancer cell growth, angiogenesis, and invasion. ω-3 PUFAs may act by suppressing the metabolism of arachidonic acid to form proinflammatory mediators or as a precursors of novel lipid mediators with pro-resolving activity, while COXIBs are able to modulate inflammatory response by inhibiting cyclooxygenase 2 (COX-2), an inducible prostaglandin synthase overexpressed in several human cancers. As recently has been postulated, the anti-inflammation and pro-resolution processes are not equivalent. A family of lipid mediators from ω-3 PUFAs can act as agonist promoting resolution, while antinflammatory agents such as COXIBs may act as antagonists limiting the inflammatory response. The present paper reviews the current knowledge about the role of PUFAs and its derivatives (metabolites), as well as the COXIBs activity in cancer process as a sinergic therapeutic alternative for cancer treatment.

Efecto del aceite de Chia rico en ácidos graso w-3 sobre el desarrollo de un adenocarcinoma de mama murino

El cáncer de mama (CM) es el tumor más frecuente entre las mujeres. Aproximadamente, el 75% de los casos son receptor de estrógeno (ER) positivo. En particular, ERα promueve el desarrollo tumoral mientras que ERβ tendría efectos anti-proliferativos. Ha sido demostrado que los acidos grasos poliinsaturados (PUFAs) y sus derivados modulan el proceso carcinogénico. Sin embargo, aún no se conocen totalmente los mecanismos que emplean estas moléculas para regular este proceso. Nos propusimos estudiar el efecto de los PUFAs ω-3 en variables biológicas implicadas en el desarrollo de un carcinoma de mama murino, tanto in vivo como in vitro. El aceite de chía es rico en ácido αlinolénico (ALA 18:3 ω-3), mientras que el de maíz es fuente de ácido linoleico (LA 18:2 ω-6). ALA y LA son precursores de eicosanoides con efectos opuestos en el CM y también producen especies reactivas del oxígeno (ROS) que pueden modular la expresión de NFκB, un factor de transcripción con un rol controversial en la carcinogénesis. Nos propusimos determinar los posibles procesos que pueden ser activados por los PUFAs ω-3 en el CM. 40 ratones BALB/c fueron alimentados con una dieta rica en aceite de chía (ChO) o aceite de maíz (CO) y se inocularon con una línea celular murina de CM, LM3. Después de 35 días, la incidencia tumoral fue mayor en los ratones alimentados con CO en comparación con los ratones alimentados con ChO (100 vs 85%, p <0,05). El peso (1,0±0,2 vs 2,2±0,2 g, p <0,05) y el volumen tumoral (4,4±0,4 vs 7,2 ± 1,0 mm3, p <0,05), así como el número de metástasis (7,4±0,8 vs 10,0±0,1, p <0.05) fueron más bajos, mientras que el tiempo de latencia del tumor (22±1 vs 15±2 d, p <0,05) fue mayor en los ratones alimentados con la dieta ChO. Se observó una disminución del número de mitosis y un mayor número de cuerpos apoptóticos, además de una mayor infiltración de linfocitos-T (células CD3+) en el grupo ChO, respecto del grupo CO (p <0,05).

Breast cancer (BC) is the most common tumour where estrogen receptor (ER) plays a key role. ERα promotes tumour growth, while ERβ has an anti-proliferative effect. It has been demonstrated that ω-3 polyunsaturated fatty acid (PUFA) consumption as well as its derivated metabolites are allowed to modulate carcinogenesis although the molecular mechanisms are not fully understood. The study was aimed to determine the possible mechanisms that are activated by dietary lipids regulating BC growth in-vivo and in-vitro. Chia oil is rich in α-linolenic acid (ALA 18:3 ω-3), while corn oil is rich in linoleic acid (LA 18:2 ω-6). ALA and LA are precursors of eicosanoid with opposite effects in BC and also generate reactive oxygen species (ROS), which could affect NFκB, a transcription factor with a controversial role in carcinogenesis. 40 BALB/c mice were fed with a diet rich in Chia Oil (ChO) or Corn Oil (CO) and inoculated with LM3, a BC murine cell line. After 35 days, tumour incidence was higher in CO-fed mice compared with ChO-fed mice (100 vs 85%, p <0.05). Tumour weight (1.0±0.2 vs 2.2±0.2 g, p <0.05) and volume (4.4±0.4 vs 7.2±1.0 mm, p <0.05) as well as metastasis number (7.4±0.8 vs 10.0±0.1, p <0.05) were lower, whereas tumour latency time (22±1 vs 15±2 d, p <0.05) was longer in ChO-fed mice. A lower number of mitosis and a higher number of apoptotic bodies besides higher T-lymphocyte infiltration was observed in ChO with respect to COgroup (p <0.05). Tumours cell membranes from ChO-fed mice showed a higher percentage of PUFAs ω-3 compared with those from CO-fed mice and generated lower amounts of ω-6 pro-inflammatory eicosanoids such as 13(S)-HODE, 15(S)HETE and 5(S)-HETE (p <0.05).