Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines.

BACKGROUND: Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy. RESULTS: In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression. CONCLUSIONS: Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome.

Development of High-Loading Trastuzumab PLGA Nanoparticles: A Powerful Tool Against HER2 Positive Breast Cancer Cells.

Background: Trastuzumab, a therapeutic monoclonal antibody directed against HER2, is routinely used to treat HER2-positive breast cancer with a good response rate. However, concerns have arisen in the clinical practice due to adverse side effects. One way to overcome these limitations is to encapsulate trastuzumab in nanoparticles to improve cytotoxic activity, increase intracellular drug concentrations, escape the immune system and avoid systemic degradation of the drug in vivo. Methods: A double emulsion method was used to encapsulate trastuzumab into poly(lactic-co-glycolic) nanoparticles, effective for their biocompatibility and biodegradability. These nanocarriers, hereafter referred to as TZPs, were characterised in terms of size, homogeneity, zeta potential and tested for their stability and drug release kinetics. Finally, the TZPs cytotoxicity was assessed in vitro on the HER2 positive SKBR3 breast cancer cell line and compared to free trastuzumab. Results: The TZPs were stable, homogeneous in size, with a reduced zeta potential. They showed higher encapsulation efficiency and drug loading, a prolonged trastuzumab release kinetics that retained its physicochemical properties and functionality. TZPs showed a stronger cytotoxicity and increased apoptosis than similar doses of free trastuzumab in the cell line analysed. Confocal microscopy and flow cytometry assessed TZPs and trastuzumab cellular uptake while Western blot evaluated downstream signalling, overall HER2 content and shedding. Conclusion: TZPs exert more robust effects than free trastuzumab via a dual mode of action: TZPs are taken up by cells through an endocytosis mechanism and release the drug intracellularly for longer time. Additionally, the TZPs that remain in the extracellular space release trastuzumab which binds to the cognate receptor and impairs downstream signalling. This is the sole modality used by free trastuzumab. Remarkably, half dose of TZPs is as efficacious as the highest dose of free drug supporting their possible use for drug delivery in vivo.

Transcriptomic Analysis of Colorectal Cancer Cells Treated with Oil Production Waste Products (OPWPs) Reveals Enrichment of Pathways of Mitochondrial Functionality.

Oil production waste products (OPWPs) derive from olive mill and represent a crucial environmental problem due to their high polyphenolic content able to pollute the ground. One option to reduce the OPWPs' environmental impact is to exploit polyphenols' biological properties. We sought to analyze the transcriptomic variations of colorectal cancer cells exposed to the OPWPs extracts and hydroxytyrosol, the major component, to recognize unknown and ill-defined characteristics. Among the top affected pathways identified by GSEA, we focused on oxidative phosphorylation in an in vitro system. Colorectal cancer HCT116 and LoVo cells treated with hydroxytyrosol or OPWPs extracts showed enhancement of the respiratory chain complexes' protein levels, ATP production and membrane potential, suggesting stimulation of mitochondrial functions. The major proteins involved in mitochondrial biogenesis and fusion events of mitochondrial dynamics were positively affected, as by Western blot, fostering increase of the mitochondrial mass organized in a network of elongated organelles. Mechanistically, we proved that PPARγ mediates the effects as they are mimicked by a specific ligand and impaired by a specific inhibitor. OPWP extracts and hydroxytyrosol, thus, promote mitochondrial functionality via a feed-forward regulatory loop involving the PPARγ/PGC-1α axis. These results support their use in functional foods and as adjuvants in cancer therapy.

Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism.

The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable 'CMS4 colorectal cancer molecular subtype' is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients' outcomes.

The miR-27a/FOXJ3 Axis Dysregulates Mitochondrial Homeostasis in Colorectal Cancer Cells.

miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.

Correction: miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

BACKGROUND: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance. METHODS: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines. RESULTS: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines. CONCLUSIONS: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.