Various Stages Of Cancer Progression Research Articles

Abstract LB033: Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells

Abstract Because of the Hippo pathway’s unique ability to restrict TAZ/YAP-TEAD in promoting regeneration, any abnormality of its core components, may promote the migration, invasion, and malignancy of cancer cells. Aberrant overexpression of YAP/TAZ-TEAD promotes tumorigenesis and is therefore considered oncogenes in many solid tumors. Drug resistance is a major factor undermining the efficacy of cancer drugs. As YAP/TAZ-TEAD complex plays a role in intrinsic and acquired resistance to various chemotherapeutic and targeted therapy drugs, there is increasing interest in combining a TEAD inhibitor with various cancer therapies. Since elevated YAP/TAZ-TEAD activity has been implicated in multiple cancer types at various stages of cancer progression and cancer types, Drugging the TEAD hydrophobic pocket, which was discovered in 2015, remains as an attractive and proven strategy to modulate the activity. Despite the progresses, only 3 small molecule TEAD inhibitors are currently being tested in Phase I clinical trials which includes: VT3989b NCT04665206 from Vivacare, IK-930b NCT05228015 from Ikena Oncology and IAG-933, NCT0485737 from the Novartis Oncology. As such, there is a need to uncover alternative TEAD inhibitors for cancer therapeutics. We report novel small molecule inhibitors that form a covalent complex with the conserved cysteine of the TEAD palmitoylation site. Compounds inhibited TEAD transcriptional target genes in cancer cells. Cellular proliferation and colony forming assay data strongly displayed cellular sensitivity in Hippo signaling altered cancer cell lines. TEAD1 dependency of gastric cancer cell lines enhance cellular sensitivity in response to small molecule inhibitors. Transcriptional profiling and RNA seq data indicate that the covalent inhibitors specifically downregulate down-stream transcriptional target genes. With our exciting key data, we are exploring multiple other oncology targets to use our Pan-TEAD inhibitors either as a monotherapy or combination therapy. We expect to complete our Technology Disclosure on additional targets before the meeting and to add exciting data for the Cancer drug development community. Citation Format: Ramesh Kumar, Joel Toh, Joanne Thian, Noorul Farzana Mohideen, Jialin Sun, Sayan Chakraborty, Jayantha Gunaratne, Wanjin Hong. Identification of small molecule Pan-TEAD inhibitors disrupting YAP-TEAD protein-protein interaction and targeting gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB033.

Proteinase-activated Receptor 2: Springboard of Tumors.

Proteinase-activated receptors (PARs) were discovered more than 25 years ago and since then, their role in cancer has been under investigation. Research has primarily focused on the receptors located on the membrane of cancer cells and their impact on metabolism, intracellular signalling, and proliferation. Regarding the host response to cancer, studies have predominantly examined the relationship of thrombin receptors (PAR-1, PAR-3, and PAR-4) with blood clotting in distant metastatic spread. However, limited studies have examined the role of PARs, especially PAR-2, in the host anti-tumor immunity. This review article provides insights into the role of PAR-2 on cancer cells and immune competent cells involved in cancer development and progression. It also discussed the current knowledge of the importance of PAR-2 activation at various stages of cancer progression and its association with cancer-related pain.

Multiple programmed cell death patterns and immune landscapes in bladder cancer: Evidence based on machine learning and multi-cohorts.

Bladder cancer (BLCA) is the most prevalent malignant neoplasm of the urinary tract, and ranks seventh as the most frequent systemic neoplasm in males. Dysregulation of programmed cell death (PCD) has been implicated in various stages of cancer progression, including tumorigenesis, invasion, and metastasis. However, the correlation between multiple PCD modes and BLCA is lacking. Thus, a risk prediction model was built based on 12 models of PCD to predict prognosis and immunotherapy response in patients with BLCA. The RNA sequencing transcriptome data of BLCA were collected from the Cancer Genome Atlas Program (TCGA) and GEO datasets. Univariate Cox and LASSO regression analyzes were performed to identify PCD-related genes (PCDRGs) significant for prognosis. Multivariate Cox regression analysis was used to develop a prognostic model for PCD. Survival analysis and chi-squared test were employed to analyze the survival variations between different risk groups. Univariate and multivariate Cox analyses were performed to evaluate the model as an independent prognostic predictor. A nomogram was formulated using both clinical data and the model to predict the survival rates of BLCA patients. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were performed to analyze and elucidate the molecular mechanisms and pathways operating within different risk score groups. Furthermore, the immune landscape was investigated and the efficacy of various anti-tumor drugs was evaluated for BLCA. Finally, consensus clustering analysis was adopted to explore the association between different PCD clusters and clinical characteristics. Assessment of the public datasets and multivariate Cox analysis yielded 1254 PCDRGs, of which 10 PCDRGs for BLCA were identified. Based on the PCDRGs, a prognostic model was built for BLCA patient prognosis. Compared with the low-risk group, the high-risk group had a poorer prognosis. The model predicted area under the curve (AUC) values of 0.751, 0.753, and 0.763, respectively, for 1-, 3-, and 5-year survival of BLCA patients. The nomogram further demonstrated the credibility of the prognosis model. The low-risk group patients exhibited lower TIDE scores and higher TMB scores, implying better response of the low-risk group to immunotherapy. The consensus clustering analysis indicated that compared with PCD cluster A, PCD cluster B was significantly more expressed in PCDRGs, suggesting a closer relation of PCD cluster B to PCDRGs. Patients in PCD cluster B had lower risk scores. To summarize, the effects of 12 PCD patterns on BLCA were synthesized and the correlation between PCD and BLCA was explored. These findings provide new and convincing evidence for individualized treatment of BLCA, and help guide the treatment strategy and improve the prognosis of BLCA patients.

ECM stiffness-regulated exosomal thrombospondin-1 promotes tunneling nanotubes-based cellular networking in breast cancer cells

Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging that may be further fine-tuned by the microenvironmental changes. Extracellular matrix (ECM) stiffening due to excess deposition and crosslinking of collagen is one of the crucial tumor-microenvironmental changes that influence a plethora of cellular processes, including cell-cell communication. We herein studied the crosstalk between exosomes and tunneling nanotubes (TNT), the two distinct means of cell-cell communication under varying ECM-stiffness conditions. We show that exosomes promote the formation of tunneling nanotubes in breast cancer cells, which results in cellular internet. Interestingly, exosomes drastically increased the fraction of cells connected by TNT; however, they elicited no effect on the number of TNTs per pair of connected cells or the length of TNT. The observed pro-TNT effects of exosomes were found to be ECM-stiffness dependent. ECM-stiffness tuned exosomes were found to promote TNT formation predominantly via the ‘cell dislodgment model’. At the molecular level, exosomal thrombospondin-1 was identified as a critical pro-TNT factor. These findings underline the influence of ECM stiffening on two diverse modes of cell communication and their interdependence, which may have significant implications in cancer biomedical research.

Targeting extracellular Hsp90: A unique frontier against cancer.

The molecular chaperone Heat Shock Protein-90 (Hsp90) is known to interact with over 300 client proteins as well as regulatory factors (eg. nucleotide and proteins) that facilitate execution of its role as a chaperone and, ultimately, client protein activation. Hsp90 associates transiently with these molecular modulators during an eventful chaperone cycle, resulting in acquisition of flexible structural conformations, perfectly customized to the needs of each one of its client proteins. Due to the plethora and diverse nature of proteins it supports, the Hsp90 chaperone machinery is critical for normal cellular function particularly in response to stress. In diseases such as cancer, the Hsp90 chaperone machinery is hijacked for processes which encompass many of the hallmarks of cancer, including cell growth, survival, immune response evasion, migration, invasion, and angiogenesis. Elevated levels of extracellular Hsp90 (eHsp90) enhance tumorigenesis and the potential for metastasis. eHsp90 has been considered one of the new targets in the development of anti-cancer drugs as there are various stages of cancer progression where eHsp90 function could be targeted. Our limited understanding of the regulation of the eHsp90 chaperone machinery is a major drawback for designing successful Hsp90-targeted therapies, and more research is still warranted.

Breast cancer stage prediction: a computational approach guided by transcriptome analysis.

Breast cancer is the second leading cancer among women in terms of mortality rate. In recent years, its incidence frequency has been continuously rising across the globe. In this context, the new therapeutic strategies to manage the deadly disease attracts tremendous research focus. However, finding new prognostic predictors to refine the selection of therapy for the various stages of breast cancer is an unattempted issue. Aberrant expression of genes at various stages of cancer progression can be studied to identify specific genes that play a critical role in cancer staging. Moreover, while many schemes for subtype prediction in breast cancer have been explored in the literature, stage-wise classification remains a challenge. These observations motivated the proposed two-phased method: stage-specific gene signature selection and stage classification. In the first phase, meta-analysis of gene expression data is conducted to identify stage-wise biomarkers that were then used in the second phase of cancer classification. From the analysis, 118, 12 and 4 genes respectively in stage I, stage II and stage III are determined as potential biomarkers. Pathway enrichment, gene network and literature analysis validate the significance of the identified genes in breast cancer. In this study, machine learning methods were combined with principal component and posterior probability analysis. Such a scheme offers a unique opportunity to build a meaningful model for predicting breast cancer staging. Among the machine learning models compared, Support Vector Machine (SVM) is found to perform the best for the selected datasets with an accuracy of 92.21% during test data evaluation. Perhaps, biomarker identification performed here for stage-specific cancer treatment would be a meaningful step towards predictive medicine. Significantly, the determination of correct cancer stage using the proposed 134 gene signature set can possibly act as potential target for breast cancer therapeutics.

Targeting type I collagen for cancer treatment

Collagen is the most abundant protein in animals. Interactions between tumor cells and collagen influence every step of tumor development. Type I collagen is the main fibrillar collagen in the extracellular matrix and is frequently upregulated during tumorigenesis. The binding of type I collagen to its receptors on tumor cells promotes tumor cell proliferation, epithelial-mesenchymal transition and metastasis. Type I collagen also regulates the efficacy of tumor therapies, such as chemotherapy, radiotherapy and immunotherapy. Furthermore, type I collagen fragments are diagnostic markers of metastatic tumors and have prognostic value. Inhibition of type I collagen synthesis has been reported to have antitumor effects in animal models. However, collagen has also been shown to possess antitumor activity. Therefore, the roles that type I collagen plays in tumor biology are complex and tumor type-dependent. In this review, we discuss the expression and regulation of synthesis of type I collagen, as well as the role upregulated type I collagen plays in various stages of cancer progression. We also discuss the role of collagen in tumor therapy. Finally, we highlight several recent approaches targeting type I collagen for cancer treatment.

Therapeutic Potential of Certain Terpenoids as Anticancer Agents: A Scoping Review.

Simple SummaryTerpenoids are bioactive compounds with a variety of pharmacological activities, including anticancer activity. This review paper aimed to focus on the anticancer activities of certain terpenoids, including their molecular mechanism, signaling pathways and clinical trials. In addition, this review provides insight and future directions to the development of some terpenoids as potential anticancer agents.Cancer is a life-threatening disease and is considered to be among the leading causes of death worldwide. Chemoresistance, severe toxicity, relapse and metastasis are the major obstacles in cancer therapy. Therefore, introducing new therapeutic agents for cancer remains a priority to increase the range of effective treatments. Terpenoids, a large group of secondary metabolites, are derived from plant sources and are composed of several isoprene units. The high diversity of terpenoids has drawn attention to their potential anticancer and pharmacological activities. Some terpenoids exhibit an anticancer effect by triggering various stages of cancer progression, for example, suppressing the early stage of tumorigenesis via induction of cell cycle arrest, inhibiting cancer cell differentiation and activating apoptosis. At the late stage of cancer development, certain terpenoids are able to inhibit angiogenesis and metastasis via modulation of different intracellular signaling pathways. Significant progress in the identification of the mechanism of action and signaling pathways through which terpenoids exert their anticancer effects has been highlighted. Hence, in this review, the anticancer activities of twenty-five terpenoids are discussed in detail. In addition, this review provides insights on the current clinical trials and future directions towards the development of certain terpenoids as potential anticancer agents.

Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis.

Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.

Impact of Galectins in Resistance to Anticancer Therapies.

Galectins are an endogenous family of β-galactoside-binding proteins that play complex and multifaceted roles at various stages of cancer progression, including modulation of tumor cell proliferation, signaling, adhesion, migration, invasion, epithelial-mesenchymal transition, angiogenesis, and immune escape. Recently, galectins have been implicated as major therapeutic determinants that confer sensitivity or resistance to a wide range of anticancer modalities including chemotherapy, radiotherapy, targeted therapies, antiangiogenic therapies, and immunotherapies. Here, we present an integrated approach to the pleiotropic functions of galectins and discuss their emerging roles with respect to mechanisms of resistance or sensitivity to anticancer therapies. Taken together, these findings suggest that targeting galectins and/or their glycosylated ligands may help to overcome resistance and to increase the clinical efficacy of anticancer strategies.

Glypican-1 Level Is Elevated in Extracellular Vesicles Released from MC38 Colon Adenocarcinoma Cells Overexpressing Snail.

The transcription factor Snail triggers epithelial-to-mesenchymal transition (EMT), endowing cancer cells with invasive properties during tumor progression. Extracellular vesicles (EVs) released from cancer cells at various stages of cancer progression are known to influence the tumor pre-metastatic niche and metastatic potential. The aim of this study was to analyze the effect of Snail on murine colon adenocarcinoma cells (MC38 line) and on the characteristics of their EVs. Stable clones of Snail-overexpressing MC38 cells were investigated in vitro versus Mock cells. Increased expression of matrix metalloproteinase MMP-14 and augmented activity of MMP-9 and -14 were observed in Snail-MC38 cells. There was no change in the transcriptomic profile of proteoglycans in Snail-MC38 cells; however, the protein level of Glypican-1 (GPC1) was enhanced in EVs released from those cells. Our finding that GPC1 protein level was enhanced in EVs released from MC38 cells that overexpressed Snail and were in an early EMT stage might explain the specificity of the GPC1 biomarker in colon cancer diagnosis. Further, our data suggest that Snail, by changing the level of GPC1 on EVs released by colon cancer cells, may affect the generation of a distant premetastatic niche and metastatic organotropism in colon adenocarcinoma.

High-Frequency Ultrasonic Forceps for the In Vivo Detection of Cancer During Breast-Conserving Surgery

AbstractA major aim in the surgical management of soft tissue cancers is to detect and remove all cancerous tissues while ensuring noncancerous tissue remains intact. Breast-conserving surgery provides a prime illustration of this aim, since remaining cancer in breast margins results in multiple surgeries, while removal of too much unaffected tissue often has undesirable cosmetic effects. Similarly, resection of benign lymph nodes during sentinel lymph node biopsy can cause deleterious health outcomes. The objective of this study was to create an intraoperative, in vivo device to address these challenges. Instant diagnostic information generated by this device could allow surgeons to precisely and completely remove all malignant tissue during the first surgery. Surgical forceps based on Martin forceps were instrumented at the tips with high-frequency ultrasonic transducers composed of polyvinylidene difluoride, a thickness-sensing rotary potentiometer at the base, and a spring to provide the appropriate restoring force. Transducer wires within the forceps were connected to an external high-frequency pulser-receiver, activating the forceps' transmitting transducer at 50 MHz and amplifying through-transmission signals from the receiving transducer. The forceps were tested with tissue-mimicking agarose phantoms embedded with 58–550 μm polyethylene microspheres to simulate various stages of cancer progression and to provide a range of measurement values. Results were compared with measurements from standard 50 MHz immersion transducers. The results showed that the forceps displayed similar sensitivity for attenuation and increased accuracy for wave speed. The forceps could also be extended to endoscopes and laparoscopes.

Breast Cancer-Derived Exosomes Reflect the Cell-of-Origin Phenotype.

A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell-of-origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway-enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial-mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell-derived exosomes reflect the phenotype of the cells-of-origin.

Challenges and Insights in Regulation of p53 and NF‐kappaB Transcription Factors: Making the Case for Cancer Prevention from the Environmental‐Physiological Paradigm

The American Cancer Society currently reports that 42% of cancer cases are caused by avoidable risk factors, such as tobacco smoking, low fiber diets and HPV, while the remaining 58% of cases have been correlated with chronic exposure to known and unknown environmental carcinogens and endogenous inflammatory reactive oxygen species. Given the high correlation of acquired mutations in the TP53 (tumor suppression) gene in various stages of cancer progression and the associated misregulation of NF-kappaB (innate immunity and inflammation), there have been numerous efforts to deduce the mechanism of transcriptional regulation of the two families that are also associated with cell cycle regulation, cell proliferation, apoptosis, metabolism, embryonic development and somatic cell differentiation. However, there is no definitive model of p53 or NF-kappaB regulation in the nucleus that can explain how the numerous genes involved in complex cell processes are activated/repressed. The main challenges stem from the sheer structural complexity of each family member, having multiple isoforms, splice variants and post-translational modifications that subsequently interact with different epigenetic and genetic cofactors. We have reported the first crystallographic and biochemical evidence showing the structure-function relationships of key DNA-binding features and regulatory domains in p73 and RelA that play a role in gene activation and repression via DNA response element (RE) sequence recognition. While initial studies show p53 and NF-kappaB family members binding to regulatory regions for hundreds of different genes at canonical DNA RE sequences, there may be a permissive modality in sequence recognition implying the larger importance of epigenetic regulation in tumor suppression or epigenetic mis-regulation in oncogenesis. As transcription is largely regulated in response to environmental stimuli via signaling cascades, the same endogenous factors involved in regulating inflammation, wound healing and cell differentiation, e.g. gut-derived serotonin, may be key to counteracting exogenous factors in cancer onset and progression. Support or Funding Information Molecular Biophysics Training Grant, NIH Grant T32 GM008326; Ruth L. Kirschstein National Research Service Award #T32 CA009523; Diffraction data were collected at BL7-1 of the Stanford/Stanford Synchrotron Radiation Lightsource supported by the Department of Energy and National Institutes of Health [P41RR001209] Comparison of the DNA-binding residues of five structures of the p73 DBD in complex with DNA consensus sequences associated with cell-cycle arrest or apoptosis. Abdul S. Ethayathulla et al. J. Biol. Chem. 2013;288:4744–4754 DNA-binding constants of the p73 S139F DBD mutant and the ΔNp73δ isoform for GGGCA and GAACA sequences associated with cellcycle arrest and apoptosis response elements, respectively. Ciribilli et al. Nucleic Acids Res. 2013 Oct; 41(18): 8637–8653. [Figure and data generated by HT Nguyen and A Ramos] Two RelA homodimers bind tandem κB sites of E-selectin promoter asymmetrically. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 [Apo] activation domain of RelA inhibits DNA binding. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 Tandem κB sites act synergistically to activate transcription by RelA. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differentiation between melanoma cells from various stages of cancer progression

Differentiation between melanoma cells from various stages of cancer progression

Mitochondrial Redox Signaling and Tumor Progression.

Cancer cell can reprogram their energy production by switching mitochondrial oxidative phosphorylation to glycolysis. However, mitochondria play multiple roles in cancer cells, including redox regulation, reactive oxygen species (ROS) generation, and apoptotic signaling. Moreover, these mitochondrial roles are integrated via multiple interconnected metabolic and redox sensitive pathways. Interestingly, mitochondrial redox proteins biphasically regulate tumor progression depending on cellular ROS levels. Low level of ROS functions as signaling messengers promoting cancer cell proliferation and cancer invasion. However, anti-cancer drug-initiated stress signaling could induce excessive ROS, which is detrimental to cancer cells. Mitochondrial redox proteins could scavenger basal ROS and function as “tumor suppressors” or prevent excessive ROS to act as “tumor promoter”. Paradoxically, excessive ROS often also induce DNA mutations and/or promotes tumor metastasis at various stages of cancer progression. Targeting redox-sensitive pathways and transcriptional factors in the appropriate context offers great promise for cancer prevention and therapy. However, the therapeutics should be cancer-type and stage-dependent.

Abstract AS23: Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis

Abstract The cross-talk between ovarian cancer cells with the microenvironment of the site of metastasis is an essential determinant of successful metastatic colonization. microRNAs have been well established to play critical roles in various stages of cancer progression, including metastasis. However, the role of the signals from the microenvironment in regulating key microRNAs in metastasizing cancer cells has not been studied. Using a 3D culture model mimicking the human omentum, one of the principal sites of ovarian cancer metastasis, we have identified the microenvironment-induced downregulation of a tumor suppressor microRNA miR-193b in the metastasizing ovarian cancer cells. The direct interaction of the ovarian cancer cells with the mesothelial cells covering the surface of the omentum caused DNA methyltransferase 1 (DNMT1) mediated methylation and decreased expression of miR-193b. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate in the omentum both in vitro and in a mouse xenograft model of ovarian cancer metastasis. The functional effects of miR-193b were mediated at least partly through the concomitant increased expression of its target urokinase-type plasminogen activator (uPA). Our findings link paracrine signals from the microenvironment with the regulation of a key microRNA in cancer cells that is essential for the initial steps of ovarian cancer metastatic colonization. Targeting miR-193b would be a promising approach to treat ovarian cancer metastasis. Citation Format: AK Mitra, CY Chiang, P Tiwari, ME Peter, E Lengyel. Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS23.

Differentiation between single bladder cancer cells using principal component analysis of time-of-flight secondary ion mass spectrometry.

Time-of-flight-secondary ion mass spectrometry (TOF-SIMS) mass spectra measurements combined with an appropriate sample preparation protocol are the powerful tools to obtain unique information about the chemical composition of biological materials. In our studies, two questions were addressed, i.e., whether it is possible to develop a fixative-based sample preparation protocol and whether it allows one to distinguish between cells originating from various stages of cancer progression. Therefore, four human bladder cancer cell lines (with distinct malignancy degree) have been investigated. A chemical fixation protocol has been used for TOF-SIMS measurements, and mass spectra were obtained using a Bi3(+) primary ion beam. The principal component analysis (PCA) has been applied to analyze the whole range of mass spectra (without preselection of any particular masses) using two approaches of data preprocessing, namely, mean centering and autoscaling. The PC3 versus PC2 plot has showed significant differences between nonmalignant cancer cells and the cancerous ones for both of preprocessing approaches. The analysis of mass spectra of human bladder cells allows one to find a list of mass peaks with intensities significantly larger in cancerous bladder cells compared to nonmalignant cell cancer of the ureter (HCV29 cells). These findings show that TOF-SIMS in combination with PCA can be used to identify reference, human bladder cells from cancerous ones.

A Twist1-MLL-WDR5-HOTTIP Complex Regulates HOXA9 Chromatin to Facilitate Metastasis of Prostate Cancer

The Twist1 transcription factor induces a transcriptional program resulting in cellular phenotypic changes, the epithelial-mesenchymal transition (EMT), during cancer progression. Twist1 is overexpressed in prostate cancer (PCa) specimens correlating with metastatic disease. We have demonstrated previously that Twist1 activates transcription of HOXA9 which contributes to the induction of a Twist1-dependent metastatic phenotype in PCa. Twist1 is canonically known as a transcription factor, but recent data suggest an epigenetic role for Twist1 in gene regulation. During embryogenesis, the histone methyltransferase complex, MLL-WDR5 and the lncRNA HOTTIP, regulate the expression of the HOXA cluster of genes by H3K4 chromatin methylation. Interestingly, PCa whole exome sequencing has shown frequent mutations in MLL2. Taken together, we hypothesized that Twist1 cooperates with the MLL-WDR5-HOTTIP (MWH) complex to drive expression of HOXA9 mediating PCa metastasis. Isogenic PCa cell lines using parental Myc-CaP and PC3 cells were created. Stable overexpression and knockdown (KD) experiments were performed with retroviral vectors. Transient KD with siRNA used commercial liposomal transfection reagents. Molecular biology procedures such as co-immunoprecipitation (IP), RNA IP (RIP), qPCR, Western and chromatin-IP (ChIP) were performed as stated below. We also used in vitro assays that mimic the various stages of cancer progression to metastasis as below. The MSKCC cBioPortal was used to perform PCa expression analysis. Twist1 could interact with components of the MWH complex as shown by Twist1 co-IP of WDR5 and RIP of HOTTIP. Twist1 overexpression also upregulated expression of the lncRNA HOTTIP in PCa cells. Functionally, WDR5 KD reduced cellular migration and invasion in Twist1 overexpressing cells. Similarly, KD against the lncRNA component HOTTIP also reduced Twist1-induced migration. Importantly, WDR5 or HOTTIP knockdown could abrogate Twist1-induced HOXA9 expression as shown by reduced HOXA9 message and protein levels. ChIP analysis of the HOXA9 promoter revealed that Twist1-overexpressing PCa cells had increased H3K4me3 levels specific for MWH activity. Last, TWIST1and genes encoding MWH complex components were co-overexpressed in PCa samples. Twist1 plays key roles during development and is a master transcriptional regulator of the EMT that promotes cancer metastasis. We demonstrated three important findings in Twist1-overexpressing PCa: (1) Twist1 forms a complex with the MWH methyltransferase complex; (2) the MWH complex activates HOXA9 expression by H3K4me3 chromatin modification of HOXA9 promoter region; and (3) the MWH complex is required for full Twist1-induced pro-metastatic behaviors.

Returning home: psychosocial care during the re-entry phase of cancer survivorship in rural Australia.

The purpose of this research was to highlight gaps in formal psychosocial care for cancer survivors in rural communities. The study was conducted in rural Victoria, Australia, and involved interviews with people with various stages of cancer progression, cancer diagnoses and survival times, who were interviewed about their experiences of psychosocial cancer care. Interviews focused on their experience of psychosocial care and were audio-recorded and transcribed verbatim. Data were thematically analysed to identify key themes. Findings demonstrate that psychosocial care is essentially informal within the re-entry period after cancer diagnosis and treatment. Despite current Australian clinical guidelines on psychosocial care for people with cancer, which indicate the need for the provision of formal psychosocial care, participants in this study largely cared for themselves, or received informal support from family, friends and community members. Many psychosocial needs remained unmet and professional support was lacking. While this study was conducted in rural Australia, many of our findings have been echoed in studies from other countries. The findings have implications for treatment protocols and discharge planning, health professional-patient-family relationships, and the long-term well-being of cancer survivors living in rural communities. A model for understanding the experience of formal supportive care during the re-entry phase of survivorship is proposed.