Cancer stem cells in head and neck tumors: evidence for metastatic spread and treatment resistance

The major challenge in the management of patients with oral squamous cell carcinomas (OSCC) is the development of resistance to therapy leading to disseminated disease. Since cancer stem cells (CSC) have emerged as important players in OSCC metastasis, our objectives were to explore the implications of CSC in OSCC tumor progression, invasion and response to conventional therapies. Methods: A panel of well-characterized cell lines originated from the most common sites in the head and neck area was used. Cells were cultured as floating spheres or under normal adherent conditions and analyzed for CD44, ALDH, CD24, CD29, CD56 by flow cytometry, PCR arrays for genes related to stemness, metastasis and EMT . We also investigated sLeX expression, known to play a key-role in many cancers metastasis by promoting tumor cells binding to endothelial E-selectin. We analyzed the tumorigenic potential of OSCC cells by invasion assays and in vivo OSCC experimental models comparatively to CSC cells. Moreover resistance to cisplatin and radiation was assessed by annexin V/PI assay and by colony forming assay. Results: The highest levels of sLeX expression were found in cell lines originated from oral cavity (9%-47%) compared to other head and neck locations (0.1%-7%). Cells grown as spheres were 95-100% positive for sLeX compared to 10-40% of adherent counterpart. Although sLeX+ and sLeX- cells were both able to form spheres, sLeX+ spheres were predominant and larger. Flow cytometry and PCR arrays indicated that the spheres were highly enriched in CSC and metastatic markers. Consistently, the spheres showed increased invasive and tumorigenic potential, and resistance to conventional chemotherapy and radiations. Conclusion: these studies are the first to unveil a novel link between sLeX expression, stem cell formation and metastatic spread in OSCC, and provide supportive evidence for CSC resistance to treatment. Understanding the mechanisms of tumor invasion and metastasis will improve patient outcome and survival

Methylation and epigenetic modification by 5’ azacytidine and valproic acid treatment increase stemness attributes in bone sarcoma cell lines

Bone sarcoma is an aggressive malignancy with high mortality rate. Despite recent advances, the prognosis is still extremely poor. Bone sarcomas contain a small cell population with stem cell like properties, referred to as cancer stem cells (CSCs) expressing CD133 (Tirino et al, 2009; 2011). The biological relevance and regulatory mechanism of CD133 expression are not yet understood. The aim of this study is to elucidate mechanisms regulating aberrant expression of CD133 and stemness phenotype. Saos-2, MG63 and BS15 cell lines were treated with 0,5 mM valproic acid (VPA) and 3μM 5’azacytidine (5-AZA) for 48 hours alone and in combination. CD133 and stemness markers expression including OCT4, Sox2 and Nanog were analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cell number. After treatment with 5-AZA or VPA, the expression level of CD133 mRNA as well as of protein was significantly increased in all three cell lines. Also OCT4, Sox2 and Nanog, stemness markers, and vimentin, mesenchymal marker resulted to be upregulated after treatment by real time-PCR. On the contrary, the expression level of osteocalcin remained similar before and after treatment. Interestingly, combined treatment with 5-AZA and VPA induced an increase of CD133 expression in a synergistic manner in all three cell lines. In addition, sarcospheres formation rate was increased after drug treatment compared to untreated cells. Also in this case, the drug combination lead to synergistic increase of formation rate of spheres. In conclusion, our results indicate that DNA methylation is an important determinant of CD133 and stemness profile in human bone sarcomas and this mechanism may be associated with histone deacetilase inhibition

The role of autophagy in resistance to targeted therapies

Autophagy is a self-degradative cellular process, involved in stress response such as starvation, hypoxia, and oxidative stress. This mechanism balances macro-molecule recycling to regulate cell homeostasis. In cancer, autophagy play a role in the development and progression, while several studies describe it as one of the key processes in drug resistance. In the last years, in addition to standard anti-cancer treatments such as chemotherapies and irradiation, targeted therapy became one of the most adopted strategies in clinical practices, mainly due to high specificity and reduced side effects. However, similar to standard treatments, drug resistance is the main challenge in most patients. Here, we summarize recent studies that investigated the role of autophagy in drug resistance after targeted therapy in different types of cancers. We highlight positive results and limitations of pre-clinical and clinical studies in which autophagy inhibitors are used in combination with targeted therapies. Refereed/Peer-reviewe

Hyaluronan-Based Gel Promotes Human Dental Pulp Stem Cells Bone Differentiation by Activating YAP/TAZ Pathway.

Background: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. Methods: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. Results: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. Conclusions: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells. Keywords: YAP/TAZ pathway; dental pulp stem cells; hyaluronic acid; osteogenic differentiation

Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration

Human DPSCs fabricate vascularized woven bone tissue : a new tool in bone tissue engineering

Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells that have been successfully used in human bone tissue engineering. To establish whether these cells can lead to a bone tissue ready to be grafted, we checked DPSCs for their osteogenic and angiogenic differentiation capabilities with the specific aim of obtaining a new tool for bone transplantation. Therefore, hDPSCs were specifically selected from the stromal-vascular dental pulp fraction, using appropriate markers, and cultured. Growth curves, expression of bone-related markers, calcification and angiogenesis as well as an in vivo transplantation assay were performed. We found that hDPSCs proliferate, differentiate into osteoblasts and express high levels of angiogenic genes, such as vascular endothelial growth factor and platelet-derived growth factor A. Human DPSCs, after 40 days of culture, give rise to a 3D structure resembling a woven fibrous bone. These woven bone (WB) samples were analysed using classic histology and synchrotron-based, X-ray phase-contrast microtomography and holotomography. WB showed histological and attractive physical qualities of bone with few areas of mineralization and neovessels. Such WB, when transplanted into rats, was remodelled into vascularized bone tissue. Taken together, our data lead to the assumption that WB samples, fabricated by DPSCs, constitute a noteworthy tool and do not need the use of scaffolds, and therefore they are ready for customized regeneration

A genome-wide perspective about the diversity and demographic history of seven Spanish goat breeds

Altres ajuts: Félix Goyache is supported by Grant FICYT GRUPIN14-113. Valentin Balteanu is the recipient of a grant awarded under the frame of the European Social Fund, Human Resources Development Operational Program 2007-2013, Project No. POSDRU/159/1.5/S/132765.Background: The main goal of the current work was to infer the demographic history of seven Spanish goat breeds (Malagueña, Murciano-Granadina, Florida, Palmera, Mallorquina, Bermeya and Blanca de Rasquera) based on genomewide diversity data generated with the Illumina Goat SNP50 BeadChip (population size, N = 176). Five additional populations from Europe (Saanen and Carpathian) and Africa (Tunisian, Djallonké and Sahel) were also included in this analysis (N = 80) for comparative purposes. Results: Our results show that the genetic background of Spanish goats traces back mainly to European breeds although signs of North African admixture were detected in two Andalusian breeds (Malagueña and MurcianoGranadina). In general, observed and expected heterozygosities were quite similar across the seven Spanish goat breeds under analysis irrespective of their population size and conservation status. For the Mallorquina and Blanca de Rasquera breeds, which have suffered strong population declines during the past decades, we observed increased frequencies of large-sized (ROH), a finding that is consistent with recent inbreeding. In contrast, a substantial part of the genome of the Palmera goat breed comprised short ROH, which suggests a strong and ancient founder effect. Conclusions: Admixture with African goats, genetic drift and inbreeding have had different effects across the seven Spanish goat breeds analysed in the current work. This has generated distinct patterns of genome-wide diversity that provide new clues about the demographic history of these populations

Pentose phosphate pathway inhibition induce Endoplasmic Reticulum stress and autophagy

Pentose phosphate pathway (PPP) is a major glucose catabolism pathway that supplies the cell with a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose-5-phosphate. NADPH is necessary for the detoxification of reactive oxygen species (ROS) and reductive biosynthesis. A key player in this pathway is the enzyme glucose-6-phosphate dehydrogenase (G6PD) that reduces NADP+ to NADPH, oxidizes glucose-6-phosphate and prevents ROS accumulation. Here, we show that the natural molecule 3,4’,5-trihydroxystilbene-3-β-d-glucoside (Polydatin) inhibits glucose-6-phosphate dehydrogenase (G6PD). As expected, G6PD inhibition causes an imbalance in NADP+/NADPH ratio, leading to a redox imbalance, followed by Endoplasmic Reticulum (ER) stress, autophagy, cell cycle block and apoptosis. we have demonstrated a link between G6PD inhibition and ER stress, showing that Unfolded Protein Response mediator such as PERK and IRE-1 have a key role in inducing autophagy and apoptosis after PPP block. Moreover, combination of PPP inhibition with autophagy inhibitors, such as chloroquine, strongly potentiate cytotoxicity on cancer cells, evidencing the role of autophagy as an escaping mechanism. This results shows that double inhibition of PPP and autophagy may be an affective therapeutic strategy against cancer

Human adipose stem cell differentiation is highly affected by cancer cells both in vitro and in vivo: implication for autologous fat grafting

Recent studies showed that mesenchymal stem cells derived from adipose tissue can promote tumour progression, raising some concerns regarding their use in regenerative medicine. In this context, we co-cultured either SAOS2 osteosarcoma or MCF7 breast cancer cells with human adipose stem cells (hASCs), in order to evaluate potential effects of cancer cells on hASCs differentiation, in vitro and in vivo. In this study we observed that both SAOS2 and MCF7 cell lines induced an increase in hASCs proliferation, compared to hASCs alone, but, surprisingly, neither changes in the expression of CD90, CD29, CD324 and vimentin, nor variations in the Twist and Slug mRNAs were detectable. Noteworthy, SAOS2 and MCF7 cells induced in hASCs an upregulation of CD34 expression and Stemness genes, including OCT3/4, Nanog, Sox2 and leptin, and a decrease in angiogenic factors, including CD31, PDGFα, PDGFRα, PDGFRβ and VEGF. SMAD and pSMAD2/3 increased only in hASCs alone. After 21 days of co-culture, hASCs differentiated both in adipocytes and endothelial cells. Moreover, co-injection of MCF7 cells with hASCs led to the formation of a highly vascularized tumour. Taken together our findings suggest that mesenchymal stem cells, under tumour cell induction, do not differentiate in vitro or facilitate the angiogenesis of the tumour in vivo, thus opening interesting new scenarios in the relationship between cancer and stem cells. These findings may also lead to greater caution, when managing autologous fat grafts in cancer patients

Polydatin (3,4’,5-trihydroxystilbene-3-β-d-glucoside) is a new inhibitor of glucose-6-phosphate dehydrogenase affecting cancer metabolism and producing a strong cytotoxic and antimetastatic effect

Polydatin (3,4’,5-trihydroxystilbene-3-β-d-glucoside) is a natural precursor of resveratrol already approved for commercialization as food supplement. It is present in many types of plants, including grape, peanut, and cassia seed where it acts as antimycotic agent. Polydatin has been proposed to have effect on cancer, including HNSCC and breast cancer, with promising results, but its mechanism of action seems to be different from resveratrol and is poorly understood. Glucose-6-phosphate dehydrogenase (G6PD) is the limiting enzyme of the pentose phosphate pathway which have been widely shown to be fundamental for tumor growth and metastasis formation. In this work our results show that polydatin inhibit G6PD in a dose dependent manner affecting cancer cell viability, causing a dose-dependent apoptosis and cell cycle arrest. Moreover, treated cells showed a strong increase of the Unfolded Protein Response (UPR), activated by a stress in the endoplasmic reticulum (ER), autophagy, reduced migration and invasion ability. Moreover, we developed a metastatic orthotopic HNSCC model in immunocompromised mice and showed that treated group had reduced tumor growth and reduce lymph nodes metastases. In conclusion here we show that Polydatin exerts a significant inhibitory effect on pentose phosphate pathway inhibiting HNSCC growth and metastases, pointing out that polydatin may be a reliable anticancer drug