Somatic mutation analyses of stem-like cells in gingivobuccal oral squamous cell carcinoma reveals DNA damage response genes.

Gingivobuccal oral squamous cell carcinoma (OSCC-GB) occurs among persons who excessively chew smokeless tobacco in India. To understand the role of cancer stem cells (CSCs) in the disease, we have performed transcriptomics analysis on RNA-seq data from OSCC-GB primary tumors. The mutational signature analysis of the identified novel and Catalogue of Somatic Mutations in Cancer (COSMIC) variants reveals DNA damage associated etiology based on identified COSMIC signatures showing a higher prevalence of C > T mutations and 1 bp T/(A) nucleotide insertions, pointing to the role of smokeless tobacco carcinogens. The differential somatic mutational, functional impact predictions, and survival analysis reveals the role of DNA damage response-related genes, with the CREBBP gene as a major player. The new CSC somatic variants identified in the study may play a crucial role in cancer metastasis, local-regional recurrence, chemo- and/or radioresistance that contributes to high mortality of the Indian OSCC-GB patients.

Human primary chronic wound derived fibroblasts demonstrate differential pattern in expression of fibroblast specific markers, cell cycle arrest and reduced proliferation.

INTRODUCTION: Although wound refers to simple cut in the skin, most wounds don't heal because of the various local and systemic factors that lead to its complexity and chronicity. Thus, prior understanding of the status of the wound is necessary and methods that can differentiate between the healing and non-healing wounds at a much earlier stage is crucial for a successful treatment. METHODS: The current study aims at differentiating Acute Wound Fibroblasts (AWFs) and Chronic Wound Fibroblasts (CWFs) based on differential expression of fibroblast specific markers such as Vimentin and Alpha Smooth Muscle Actin (α-SMA) and compare its cell cycle and proliferation. RESULTS: Immunostaining and western blotting analysis showed that, AWFs and CWFs differentially expressed vimentin and α-SMA, with AWFs and CWFs showing higher expression of vimentin and α-SMA respectively. AWFs showed higher distributions in G0/G1 (67.43% vs. 62.16%), S phase (22.61% vs. 8.51%) compared to CWFs. However, AWFs showed decreased distributions compared to CWFs in G2 + M phase (8.14% vs. 10.6%). Thus, it was observed that CWFs showed cell cycle arrest in the G1/G0 phase and inhibited DNA synthesis, which was further confirmed by reduced proliferation of CWFs. We suggest that, differential expression of the cell specific markers can be attributed to its pathophysiological status and chronicity of the wound and reduced proliferation rate of CWFs is due to lesser expression of vimentin, which is a key protein for in vitro cell proliferation. CONCLUSIONS: Outcome of the study serve as an immunological tool to guide the chronicity of the wound, which helps to understand the wound towards design of personalized care. The findings also represent a promising opportunity to gain insight into how cell cycle arrest can impact on wound healing and clinical outcomes.

Myofibroblast progeny in wound biology and wound healing studies.

Fibroblasts and myofibroblasts play a myriad of important roles in human tissue function, especially in wound repair and healing. Among all cells, fibroblasts are group of cells that decide the status of wound as they maintain tissue homeostasis. Currently, the increase in the deleterious effects of chronic wound and their morbidity rate has necessitated the need to understand the influence of fibroblasts and myofibroblasts, which chiefly originate locally from tissue-resident fibroblasts to address the same. Wound pathophysiology is complex, herein, we have discussed fibroblast and myofibroblast heterogeneity in skin and different organs by understanding the phenotypical and functional properties of each of its sub-populations in the process of wound healing. Recent advancements in fibroblast activation, differentiation to myofibroblasts, proliferation and migration are discussed in detail. Fibroblasts and myofibroblasts are key players in wound healing and wound remodelling, respectively, and their significance in wound repair is discussed. An increased understanding of their biology during wound healing also gives an opportunity to explore more of fibroblast and myofibroblast focused therapies to treat chronic wounds which are clinical challenges. In this regard, in the current review, we have described the different methods for isolation of primary fibroblasts and myofibroblasts from both animal models and humans, and their characterization. Additionally, we have also provided details on possible molecular targets for better understanding of prognosis, diagnosis and treatment of chronic wounds. Information will help both researchers and clinicians in providing molecular insight that enable them for effective chronic wound management. The knowledge of intimate dialogue between the fibroblast, sub-populations like, myofibroblast and their microenvironment, will serve useful in determining novel, efficient and specific therapeutic targets to treat pathological wound conditions.

AMP-activated protein kinase promotes epithelial-mesenchymal transition in cancer cells through Twist1 upregulation.

The developmental programme of epithelial-mesenchymal transition (EMT), involving loss of epithelial and acquisition of mesenchymal properties, plays an important role in the invasion-metastasis cascade of cancer cells. In the present study, we show that activation of AMP-activated protein kinase (AMPK) using A769662 led to a concomitant induction of EMT in multiple cancer cell types, as observed by enhanced expression of mesenchymal markers, decrease in epithelial markers, and increase in migration and invasion. In contrast, inhibition or depletion of AMPK led to a reversal of EMT. Importantly, AMPK activity was found to be necessary for the induction of EMT by physiological cues such as hypoxia and TGFß treatment. Furthermore, AMPK activation increased the expression and nuclear localization of Twist1, an EMT transcription factor. Depletion of Twist1 impaired AMPK-induced EMT phenotypes, suggesting that AMPK might mediate its effects on EMT, at least in part, through Twist1 upregulation. Inhibition or depletion of AMPK also attenuated metastasis. Thus, our data underscore a central role for AMPK in the induction of EMT and in metastasis, suggesting that strategies targeting AMPK might provide novel approaches to curb cancer spread.

Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation.

The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKß. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca(2+)/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-ß.

Actions of transforming growth factor-ß are largely context dependent. For instance, TGF-ß is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-ß also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-ß would change in the context of transformation, we have compared the differential gene regulation by TGF-ß in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-ß in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-ß is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-ß. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-ß in hFhTERT cells and TGF-ß conferred action of these cells was mediated by WNT4. While TGF-ß activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-ß which matched with the response of HT1080 cells to TGF-ß. These data suggest context dependent activation of non-canonical signaling by TGF-ß.

Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments.

Pattern Recognition-Based Approach for Identifying Metabolites in Nuclear Magnetic Resonance-Based Metabolomics.

Identification and assignments of metabolites is an important step in metabolomics and is necessary for the discovery of new biomarkers. In nuclear magnetic resonance (NMR) spectroscopy-based studies, the conventional approach involves a database search, wherein chemical shifts are assigned to specific metabolites by use of a tolerance limit. This is inefficient because deviation in chemical shifts associated with pH or temperature variations, as well as missing peaks, impairs a robust comparison with the database. We propose here a novel method based on matching the pattern of peaks rather than absolute tolerance thresholds, using a combination of geometric hashing and similarity scoring techniques. Tests with 719 metabolites from the Human Metabolome Database (HMDB) show that 100% of the metabolites can be assigned correctly when accurate data are available. A high success rate is obtained even in the presence of large chemical shift deviations such as 0.5 ppm in (1)H and 3 ppm in (13)C and missing peaks (up to 50%), compared to nearly no assignments obtained under these conditions with existing methods that employ a direct database search approach. The method was evaluated on experimental data on a mixture of 16 metabolites at eight different combinations of pH and temperature conditions. The pattern recognition approach thus helps in identification and assignment of metabolites independent of the pH, temperature, and ionic strength used, thereby obviating the need for spectral calibration with internal or external standards.

Protamine-carboxymethyl cellulose magnetic nanocapsules for enhanced delivery of anticancer drugs against drug resistant cancers.

Multidrug resistance is a major therapeutic challenge faced in the conventional chemotherapy. Nanocarriers are beneficial in the transport of chemotherapeutics by their ability to bypass the P-gp efflux in cancers. Most of the P-gp inhibitors under phase II clinical trial are facing failures and hence there is a need to develop a suitable carrier to address P-gp efflux in cancer therapy. Herein, we prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin against highly drug resistant HeLa cells. The experimental results revealed that improved cellular uptake, enhanced drug intensity profile with greater percentage of apoptotic cells was attained when doxorubicin loaded magnetic nanocapsules were used in the presence of external magnetic field. Hence, we conclude that this magnetic field assisted nanocapsule system can be used for delivery of chemotherapeutics for potential therapeutic efficacy at minimal dose in multidrug resistant cancers. FROM THE CLINICAL EDITOR: Many cancer drugs fail when cancer cells become drug resistant. Indeed, multidrug resistance (MDR) is a major therapeutic challenge. One way that tumor cells attain MDR is by over expression of molecular pumps comprising of P-glycoprotein (P-gp) and multidrug resistant proteins (MRP), which can expel chemotherapeutic drugs out of the cells. In this study, the authors prepared novel protamine and carboxymethyl cellulose polyelectrolyte multi-layered nanocapsules modified with Fe3O4 nanoparticles for the delivery of doxorubicin. The results show that there was better drug delivery and efficacy even against MDR tumor cells.

Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells.

INTRODUCTION: Matrix detachment triggers anoikis, a form of apoptosis, in most normal epithelial cells, while acquisition of anoikis resistance is a prime requisite for solid tumor growth. Of note, recent studies have revealed that a small population of normal human mammary epithelial cells (HMECs) survive in suspension and generate multicellular spheroids termed 'mammospheres'. Therefore, understanding how normal HMECs overcome anoikis may provide insights into breast cancer initiation and progression. METHODS: Primary breast tissue-derived normal HMECs were grown as adherent monolayers or mammospheres. The status of AMP-activated protein kinase (AMPK) and PEA15 signaling was investigated by immunoblotting. Pharmacological agents and an RNA interference (RNAi) approach were employed to gauge their roles in mammosphere formation. Immunoprecipitation and in vitro kinase assays were undertaken to evaluate interactions between AMPK and PEA15. In vitro sphere formation and tumor xenograft assays were performed to understand their roles in tumorigenicity. RESULTS: In this study, we show that mammosphere formation by normal HMECs is accompanied with an increase in AMPK activity. Inhibition or knockdown of AMPK impaired mammosphere formation. Concomitant with AMPK activation, we detected increased Ser116 phosphorylation of PEA15, which promotes its anti-apoptotic functions. Inhibition or knockdown of AMPK impaired PEA15 Ser116 phosphorylation and increased apoptosis. Knockdown of PEA15, or overexpression of the nonphosphorylatable S116A mutant of PEA15, also abrogated mammosphere formation. We further demonstrate that AMPK directly interacts with and phosphorylates PEA15 at Ser116 residue, thus identifying PEA15 as a novel AMPK substrate. Together, these data revealed that AMPK activation facilitates mammosphere formation by inhibition of apoptosis, at least in part, through Ser116 phosphorylation of PEA15. Since anoikis resistance plays a critical role in solid tumor growth, we investigated the relevance of these findings in the context of breast cancer. Significantly, we show that the AMPK-PEA15 axis plays an important role in the anchorage-independent growth of breast cancer cells both in vitro and in vivo. CONCLUSIONS: Our study identifies a novel AMPK-PEA15 signaling axis in the anchorage-independent growth of both normal and cancerous mammary epithelial cells, suggesting that breast cancer cells may employ mechanisms of anoikis resistance already inherent within a subset of normal HMECs. Thus, targeting the AMPK-PEA15 axis might prevent breast cancer dissemination and metastasis.

Bmi1 regulates self-renewal and epithelial to mesenchymal transition in breast cancer cells through Nanog.

BACKGROUND: The Bmi1 polycomb ring finger oncogene, a transcriptional repressor belonging to the Polycomb group of proteins plays an important role in the regulation of stem cell self-renewal and is elevated in several cancers. In the current study, we have explored the role of Bmi1 in regulating the stemness and drug resistance of breast cancer cells. METHODS: Using real time PCR and immunohistochemistry primary breast tissues were analyzed. Retro- and lentiviruses were utilized to overexpress and knockdown Bmi1, RT-PCR and Western blot was performed to evaluate mRNA and protein expression. Stemness properties were analyzed by flow cytometry and sphere-formation and tumor formation was determined by mouse xenograft experiments. Dual luciferase assay was employed to assess promoter activity and MTT assay was used to analyze drug response. RESULTS: We found Bmi1 overexpression in 64% of grade III invasive ductal breast adenocarcinomas compared to normal breast tissues. Bmi1 overexpression in immortalized and transformed breast epithelial cells increased their sphere-forming efficiency, induced epithelial to mesenchymal transition (EMT) with an increase in the expression of stemness-related genes. Knockdown of Bmi1 in tumorigenic breast cells induced epithelial morphology, reduced expression of stemness-related genes, decreased the IC50 values of doxorubicin and abrogated tumor-formation. Bmi1-high tumors showed elevated Nanog expression whereas the tumors with lower Bmi1 showed reduced Nanog levels. Overexpression of Bmi1 increased Nanog levels whereas knockdown of Bmi1 reduced its expression. Dual luciferase promoter-reporter assay revealed Bmi1 positively regulated the Nanog and NFκB promoter activity. RT-PCR analysis showed that Bmi1 overexpression activated the NFκB pathway whereas Bmi1 knockdown reduced the expression of NFκB target genes, suggesting that Bmi1 might regulate Nanog expression through the NFκB pathway. CONCLUSIONS: Our study showed that Bmi1 is overexpressed in several high-grade, invasive ductal breast adenocarcinomas, thus supporting its role as a prognostic marker. While Bmi1 overexpression increased self-renewal and promoted EMT, its knockdown reversed EMT, reduced stemness, and rendered cells drug sensitive, thus highlighting a crucial role for Bmi1 in regulating the stemness and drug response of breast cancer cells. Bmi1 may control self-renewal through the regulation of Nanog expression via the NFκB pathway.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation.

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs [EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is ~10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Inhibition of protein translation under matrix-deprivation stress in breast cancer cells.

Matrix-deprivation stress leads to cell-death by anoikis, whereas overcoming anoikis is critical for cancer metastasis. Work from our lab and others has identified a crucial role for the cellular energy sensor AMPK in anoikis-resistance, highlighting a key role for metabolic reprogramming in stress survival. Protein synthesis is a major energy-consuming process that is tightly regulated under stress. Although an increase in protein synthesis in AMPK-depleted experimentally-transformed MEFs has been associated with anoikis, the status and regulation of protein translation in epithelial-origin cancer cells facing matrix-detachment remains largely unknown. Our study shows that protein translation is mechanistically abrogated at both initiation and elongation stages by the activation of the unfolded protein response (UPR) pathway and inactivation of elongation factor eEF2, respectively. Additionally, we show inhibition of the mTORC1 pathway known for regulation of canonical protein synthesis. We further functionally assay this inhibition using SUnSET assay, which demonstrates repression of global protein synthesis in MDA-MB-231 and MCF7 breast cancer cells when subjected to matrix-deprivation. In order to gauge the translational status of matrix-deprived cancer cells, we undertook polysome profiling. Our data revealed reduced but continuous mRNA translation under matrix-deprivation stress. An integrated analysis of transcriptomic and proteomic data further identifies novel targets that may aid cellular adaptations to matrix-deprivation stress and can be explored for therapeutic intervention.

Label-free multimodal electro-thermo-mechanical (ETM) phenotyping as a novel biomarker to differentiate between normal, benign, and cancerous breast biopsy tissues.

BACKGROUND: Technologies for quick and label-free diagnosis of malignancies from breast tissues have the potential to be a significant adjunct to routine diagnostics. The biophysical phenotypes of breast tissues, such as its electrical, thermal, and mechanical properties (ETM), have the potential to serve as novel markers to differentiate between normal, benign, and malignant tissue. RESULTS: We report a system-of-biochips (SoB) integrated into a semi-automated mechatronic system that can characterize breast biopsy tissues using electro-thermo-mechanical sensing. The SoB, fabricated on silicon using microfabrication techniques, can measure the electrical impedance (Z), thermal conductivity (K), mechanical stiffness (k), and viscoelastic stress relaxation (%R) of the samples. The key sensing elements of the biochips include interdigitated electrodes, resistance temperature detectors, microheaters, and a micromachined diaphragm with piezoresistive bridges. Multi-modal ETM measurements performed on formalin-fixed tumour and adjacent normal breast biopsy samples from N = 14 subjects were able to differentiate between invasive ductal carcinoma (malignant), fibroadenoma (benign), and adjacent normal (healthy) tissues with a root mean square error of 0.2419 using a Gaussian process classifier. Carcinoma tissues were observed to have the highest mean impedance (110018.8 ± 20293.8 Ω) and stiffness (0.076 ± 0.009 kNm-1) and the lowest thermal conductivity (0.189 ± 0.019 Wm-1 K-1) amongst the three groups, while the fibroadenoma samples had the highest percentage relaxation in normalized load (47.8 ± 5.12%). CONCLUSIONS: The work presents a novel strategy to characterize the multi-modal biophysical phenotype of breast biopsy tissues to aid in cancer diagnosis from small-sized tumour samples. The methodology envisions to supplement the existing technology gap in the analysis of breast tissue samples in the pathology laboratories to aid the diagnostic workflow.

Standardized ready-to-use laboratory protocol to isolate primary human wound associated fibroblasts from infected wound samples for clinical applications.

Owing to the importance of fibroblasts in healing of wounds, it is necessary to isolate and culture them under in vitro conditions for the purpose of understanding the wound biology, drug discovery and development of personalized treatment. Although, several fibroblast cell lines are commercially available, they fail to represent the patient associated parameters. However, establishing a primary fibroblast culture, especially from infected wound samples, is challenging as the sample is more prone to contamination and number of live cells will be minimum in heterogeneous population. Also, it takes lot of efforts and resources for optimization of the protocol to get good quality cell lines from wound samples necessitating multiple trials, resulting in large number of clinical samples to be processed. To the best of our knowledge, for the first time we are reporting the standardized protocol to isolate primary human fibroblasts from acute and chronic wound samples. In this study, various parameters such as explant size (1-2 mm), explant drying time (2 min), transportation and growth culture media (antibiotics (working concentrations 1-3) and serum concentration (10%)) have been optimised. This can be altered for specific needs of cell in terms of both quality and quantity. Outcome of the work provides a ready-to-use protocol, which is very useful to those who want to initiate primary fibroblasts cell culture from infected wound samples either for clinical or research purpose. Further, these cultured primary wound associated fibroblasts have various clinical and biomedical applications in tissue grafting, treatment of burns and scars and wound regeneration especially in non-healing chronic wounds.

Species- and cell type-specific requirements for cellular transformation.

Recent evidence suggests that human cells require more genetic changes for neoplastic transformation than do their murine counterparts. However, a precise enumeration of these differences has never been undertaken. We have determined that perturbation of two signaling pathways-involving p53 and Raf-suffices for the tumorigenic conversion of normal murine fibroblasts, while perturbation of six pathways-involving p53, pRb, PP2A, telomerase, Raf, and Ral-GEFs-is needed for human fibroblasts. Cell type-specific differences also exist in the requirements for tumorigenic transformation: immortalized human fibroblasts require the activation of Raf and Ral-GEFs, human embryonic kidney cells require the activation of PI3K and Ral-GEFs, and human mammary epithelial cells require the activation of Raf, PI3K, and Ral-GEFs.

Ras modulates Myc activity to repress thrombospondin-1 expression and increase tumor angiogenesis.

Tumor angiogenesis is postulated to be regulated by the balance between pro- and anti-angiogenic factors. We demonstrate that the critical step in establishing the angiogenic capability of human cells is the repression of the critical anti-angiogenic factor, thrombospondin-1 (Tsp-1). This repression is essential for tumor formation by mammary epithelial cells and kidney cells engineered to express SV40 early region proteins, hTERT, and H-RasV12. We have uncovered the signaling pathway leading from Ras to Tsp-1 repression. Ras induces the sequential activation of PI3 kinase, Rho, and ROCK, leading to activation of Myc through phosphorylation; phosphorylation of Myc via this mechanism enables it to repress Tsp-1 expression. We thus describe a novel mechanism by which the cooperative activity of the oncogenes, ras and myc, leads directly to angiogenesis and tumor formation.

AMP-activated protein kinase promotes breast cancer stemness and drug resistance.

Breast cancer stem cells (BCSCs) are a major cause of therapy resistance and tumour progression. Currently, their regulation is not entirely understood. Previous work from our laboratory demonstrated a context-specific pro-tumorigenic role for AMP-activated protein kinase (AMPK) under anchorage-deprivation and mammosphere formation, which are hallmarks of BCSCs. Therefore, we investigated the role of AMPK in the maintenance of BCSC state/function. AMPK depletion reduces serial sphere formation in vitro and tumour initiation in vivo. Intriguingly, tumour-derived cell analysis using stem cell markers and functional assays revealed that AMPK is required for the maintenance of BCSC populations in vivo. AMPK promotes the expression of stemness genes such as NANOG, SOX2 and BMI1 through the transcriptional upregulation of TWIST via promoter acetylation. Further, AMPK-driven stemness plays a critical role in doxorubicin resistance. Significantly, AMPK activity increased after chemotherapy in patient-derived tumour samples alongside an increase in stemness markers. Importantly, AMPK depletion sensitises mouse tumours to doxorubicin treatment. Our work indicates that targeting of AMPK in conjunction with regular chemotherapy is likely to reduce the stem cell pool and improve chemosensitivity in breast cancers.

RapidET: a MEMS-based platform for label-free and rapid demarcation of tumors from normal breast biopsy tissues.

The rapid and label-free diagnosis of malignancies in ex vivo breast biopsy tissues has significant utility in pathology laboratories and operating rooms. We report a MEMS-based platform integrated with microchips that performs phenotyping of breast biopsy tissues using electrothermal sensing. The microchip, fabricated on a silicon substrate, incorporates a platinum microheater, interdigitated electrodes (IDEs), and resistance temperature detectors (RTDs) as on-chip sensing elements. The microchips are integrated onto the platform using a slide-fit contact enabling quick replacement for biological measurements. The bulk resistivity (ρ B ), surface resistivity (ρ S ), and thermal conductivity (k) of deparaffinized and formalin-fixed paired tumor and adjacent normal breast biopsy samples from N = 8 patients were measured. For formalin-fixed samples, the mean ρ B for tumors showed a statistically significant fold change of 4.42 (P = 0.014) when the tissue was heated from 25 °C to 37 °C compared to the adjacent normal tissue, which showed a fold change of 3.47. The mean ρ S measurements also showed a similar trend. The mean k of the formalin-fixed tumor tissues was 0.309 ± 0.02 W m-1 K-1 compared to a significantly higher k of 0.563 ± 0.028 W m-1 K-1 for the adjacent normal tissues. A similar trend was observed in ρ B, ρ S, and k for the deparaffinized tissue samples. An analysis of a combination of ρ B , ρ S , and k using Fisher's combined probability test and linear regression suggests the advantage of using all three parameters simultaneously for distinguishing tumors from adjacent normal tissues with higher statistical significance.

Surface-Coated Cerium Nanoparticles to Improve Chemotherapeutic Delivery to Tumor Cells.

The antioxidant property of cerium oxide nanoparticles has increased their demand as a nanocarrier to improve the delivery and therapeutic efficacy of anticancer drugs. Here, we report the synthesis of alginate-coated ceria nanoformulations (ceria NPs) and characterization using FTIR spectroscopy, Raman microscopy, and X-ray diffraction. The synthesized ceria NPs show negligible inherent in vitro toxicity when tested on a MDA-MB-231 breast cancer cell line at higher particle concentrations. Upon loading these particles with doxorubicin (Dox) and paclitaxel (PTX) drugs, we observe a potential synergistic cytotoxic effect mediated by the drug and the ceria NPs, resulting in the better killing capacity as well as suppression of cell migration against the MDA-MB-231 cell line. Further, to verify the immune-escaping capacity before targeting cancer cells, we coated the drug-loaded ceria NPs with the membrane of MDA-MB-231 cells using an extrusion method. The resultant delivery system exhibited in vitro preferential uptake by the MDA-MB-231 cell line and showed reduced uptake by the murine macrophage cell line (RAW 264.7), assigning its potential application as non-immunogenic personalized therapy in targeting and killing of cancer cells.