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Supplementary Table S2: qPCR primer for subcluster E genes

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of “mechanical fitness” in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell–macrophage cocultures revealed that macrophages promoted epithelial–mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis.Significance:The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2–M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle–related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment.Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853–64. ©2017 AACR.

Supplementary Figure S1: Time-course expression analysis of LNCaP cells reveals dynamic and heterogeneous responses to androgen stimulation. Supplementary Figure S2: Cell cycle progression of LNCaP cells treated with or without R1881 after cell synchronization by double thymidine block (DTB). Supplementary Figure S3: The summary of aligned and unaligned single-cell RNA-seq reads counts. Supplementary Figure S4: Correlation of single-cell RNA-seq ensemble reads with representative bulk RNA-seq reads. Supplementary Figure S5: Venn diagrams illustrating the comparison of differentially expressed genes found in single- and bulk-cell LNCaP experiments. Supplementary Figure S6: The Mclust algorithm defines likely subgroups using a "model-based" approach. Supplementary Figure S7: Each cell within each subcluster exhibits a similar gene expression profile. Supplementary Figure S8: Gleason score and biochemical recurrence status compared to each subcluster''s gene expression profile. Supplementary Figure S9: Differential expression of 10 genes in LNCaP''s 8.1 subpopulation of cells. Supplementary Figure S10: High single-cell expression of 10 genes in PC3 similar to 12hr-R1881 LNCaP cells.

Supplementary Materials and Methods, Figures and Tables

Bladder tumor-infiltrating CD56CD56Intratumoral CD56KLRF1 is a promising prognostic marker in bladder cancer and may guide treatment decisions upon validation.

International audience; This work investigates the thermal expansion behaviors of Fe30Co30Ni30Cr10-xMnx High Entropy Alloys (HEAs) (x = 0, 5, 10 at.%) from 400 K to 1200 K. Interestingly, comparing to that of Cantor alloy, a decrease in Cr and Mn in Co–Cr–Fe–Mn–Ni system could significantly decrease the thermal expansion coefficient by 45.2%; Fe30Co30Ni30Cr10-xMnx (x = 0, 5, 10) also showed an abrupt change in thermal expansion behaviors similar to that of Invar alloys. Experimental and theoretical analysis suggest the abrupt change in thermal expansion behaviors of these HEAs were associated with the transition from ferromagnetism to paramagnetism, and these alloys appear to exhibit the dimensional stability of the Invar effect. Since the Invar effect is related to ferromagnetic properties, the amount of Cr and Mn in the HEAs would influence the suppression of thermal expansion due to different intensity of anti-ferromagnetic coupling effect. This research contributes to the understanding of the thermal expansion behaviors of Co–Cr–Fe–Mn–Ni high entropy alloys and the effects of antiferromagnetic elements

Purpose Human innate lymphoid cells (hILCs) are lineage‐negative immune cells that do not express rearranged adaptive antigen receptors. Natural killer (NK) cells are hILCs that contribute to cancer defense. The role of non‐NK hILCs in cancer is unclear. Our study aimed to characterize non‐NK hILCs in bladder cancer. Experimental design Mass cytometry was used to characterize intratumoral non‐NK hILCs based on 35 parameters, including receptors, cytokines, and transcription factors from 21 muscle‐invasive bladder tumors. Model‐based clustering was performed on t‐distributed stochastic neighbor embedding (t‐SNE) coordinates of hILCs, and the association of hILCs with tumor stage was analyzed. Results Most frequent among intratumoral non‐NK hILCs were hILC1s, which were increased in higher compared with lower stage tumors. Intratumoral hILC1s were marked by Th17‐like phenotype with high RORγt, IL‐17, and IL‐22 compared to Th1 differentiation markers, including Tbet, perforin, and IFN‐γ. Compared with intratumoral hILC2s and hILC3s, hILC1s also had lower expression of activation markers (NKp30, NKp46, and CD69) and increased expression of exhaustion molecules (PD‐1 and Tim3). Unsupervised clustering identified nine clusters of bladder hILCs, which were not defined by the primary hILC subtypes 1–3. hILC1s featured in all the nine clusters indicating that intratumoral hILC1s displayed the highest phenotypic heterogeneity among all hILCs. Conclusions hILC1s are increased in higher stage tumors among patients with muscle‐invasive bladder cancer. These intratumoral hILC1s exhibit an exhausted phenotype and Th17‐like differentiation, identifying them as potential targets for immunotherapy., This study characterizes the diversity of tumor‐infiltrating ILCs within muscle‐invasive bladder cancer. Unique ILC subtypes were identified that differed from traditional classifications of ILCs. We further found that bladder ILC1s displayed a Th17‐like phenotype and correlated with tumor stage, suggesting further investigations into their role in cancer and potential immunotherapy targets.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of “mechanical fitness” in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell–macrophage cocultures revealed that macrophages promoted epithelial–mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. Significance: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

The interplay between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) is central to maintain energy homeostasis. It remains to be determined whether there is a mechanism governing metabolic fluxes based on substrate availability in microenvironments. Here we show that menin is a key transcription factor regulating the expression of OXPHOS and glycolytic genes in cancer cells and primary tumors with poor prognosis. A group of menin-associated proteins (MAPs), including KMT2A, MED12, WAPL, and GATA3, is found to restrain menin&rsquo, s full function in this transcription regulation. shRNA knockdowns of menin and MAPs result in reduced ATP production with proportional alterations of cellular energy generated through glycolysis and OXPHOS. When shRNA knockdown cells are exposed to metabolic stress, the dual functionality can clearly be distinguished among these metabolic regulators. A MAP can negatively counteract the regulatory mode of menin for OXPHOS while the same protein positively influences glycolysis. A close-proximity interaction between menin and MAPs allows transcriptional regulation for metabolic adjustment. This coordinate regulation by menin and MAPs is necessary for cells to rapidly adapt to fluctuating microenvironments and to maintain essential metabolic functions.

Background Epigenetic alterations are involved in most cancers, but its application in cancer diagnosis is still limited. More practical and intuitive methods to detect the aberrant expressions from clinical samples using highly sensitive biomarkers are needed. In this study, we developed a novel approach in identifying, visualizing, and quantifying the biallelic and multiallelic expressions of an imprinted gene panel associated with cancer status. We evaluated the normal and aberrant expressions measured using the imprinted gene panel to formulate diagnostic models which could accurately distinguish the imprinting differences of normal and benign cases from cancerous tissues for each of the ten cancer types. Results The Quantitative Chromogenic Imprinted Gene In Situ Hybridization (QCIGISH) method developed from a 1013-case study which provides a visual and quantitative analysis of non-coding RNA allelic expressions identified the guanine nucleotide-binding protein, alpha-stimulating complex locus (GNAS), growth factor receptor-bound protein (GRB10), and small nuclear ribonucleoprotein polypeptide N (SNRPN) out of five tested imprinted genes as efficient epigenetic biomarkers for the early-stage detection of ten cancer types. A binary algorithm developed for cancer diagnosis showed that elevated biallelic expression (BAE), multiallelic expression (MAE), and total expression (TE) measurements for the imprinted gene panel were associated with cell carcinogenesis, with the formulated diagnostic models achieving consistently high sensitivities (91–98%) and specificities (86–98%) across the different cancer types. Conclusions The QCIGISH method provides an innovative way to visually assess and quantitatively analyze individual cells for cancer potential extending from hyperplasia and dysplasia until carcinoma in situ and invasion, which effectively supplements standard clinical cytologic and histopathologic diagnosis for early cancer detection. In addition, the diagnostic models developed from the BAE, MAE, and TE measurements of the imprinted gene panel GNAS, GRB10, and SNRPN could provide important predictive information which are useful in early-stage cancer detection and personalized cancer management.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell–cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. Significance: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2–M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle–related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment. Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853–64. ©2017 AACR.

In this work, a metastable face-centered cubic High Entropy Alloy (HEA) Fe35.0Co28.7Ni24.8Cr1.1Al1.3V4.5Ti1.2Nb2.8Si0.6 was found to exhibit significantly suppressed thermal expansion coefficient (6.0 ppm/K) and stable Young's modulus over a wide temperature range. Experimental and theoretical analyses suggest that both Invar and Elinvar effects were present; in addition, the metastability of this alloy with respect to the first-order phase transition and the magnetic second-order phase transition led to abrupt changes in thermal expansion behavior. The first-order phase transition was associated with the formation of a plate-like D019 phase. The magnetic second-order phase transition decreased the Invar effect and the Elinvar effect and resulted in a significant magnetic entropy difference (−3.12 J kg−1 K−1) in this HEA of interest.

Metabolic reprogramming in cancer cells not only sustains bioenergetic and biosynthetic needs but also influences transcriptional programs, yet how chromatin regulatory networks are rewired by altered metabolism remains elusive. Here we investigate genome-scale chromatin remodeling in response to 2-hydroxyglutarate (2HG) oncometabolite using single-cell assay for transposase accessible chromatin with sequencing (scATAC-seq). We find that 2HG enantiomers differentially disrupt exquisite control of epigenome integrity by limiting α-ketoglutarate (αKG)-dependent DNA and histone demethylation, while enhanced cell-to-cell variability in the chromatin regulatory landscape is most evident upon exposure to L2HG enantiomer. Despite the highly heterogeneous responses, 2HG largely recapitulates two prominent hallmarks of the breast cancer epigenome, i.e., global loss of 5-hydroxymethylcytosine (5hmC) and promoter hypermethylation, particularly at tumor suppressor genes involved in DNA damage repair and checkpoint control. Single-cell mass cytometry further demonstrates downregulation of BRCA1, MSH2 and MLH1 in 2HG-responsive subpopulations, along with acute reversal of chromatin remodeling upon withdrawal. Collectively, this study provides a molecular basis for metabolism-epigenome coupling and identifies metabolic vulnerabilities imposed on the breast cancer epigenome.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1–STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1–STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFβ, and JAK1–STAT3 signal activation in select tumors that may be targeted by combined AXL–JAK1 inhibition. Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3β-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.

Advanced prostate cancer is a very heterogeneous disease reflecting in diverse regulations of oncogenic signaling pathways. Aberrant spatial dynamics of epidermal growth factor receptor (EGFR) promote their dimerization and clustering, leading to constitutive activation in oncogenesis. The EphB2 and Src signaling pathways are associated with the reorganization of the cytoskeleton leading to malignancy, but their roles in regulating EGFR dynamics and activation are scarcely reported. Using single-particle tracking techniques, we found that highly phosphorylated EGFR in the advanced prostate cancer cell line, PC3, was associated with higher EGFR diffusivity, as compared with LNCaP and less aggressive DU145. The increased EGFR activation and biophysical dynamics were consistent with high proliferation, migration, and invasion. After performing single-cell RNA-seq on prostate cancer cell lines and circulating tumor cells from patients, we identified that upregulated gene expression in the EphB2 and Src pathways are associated with advanced malignancy. After dasatinib treatment or siRNA knockdowns of EphB2 or Src, the PC3 cells exhibited significantly lower EGFR dynamics, cell motility, and invasion. Partial inhibitory effects were also found in DU145 cells. The upregulation of parts of the EphB2 and Src pathways also predicts poor prognosis in the prostate cancer patient cohort of The Cancer Genome Atlas. Our results provide evidence that overexpression of the EphB2 and Src signaling pathways regulate EGFR dynamics and cellular aggressiveness in some advanced prostate cancer cells.

The extraction of land cover information from remote sensing data is a complex process. Spectral information has been widely utilized in classifying remote sensing images. However, shadows limit the use of multispectral images because they result in loss of spectral radiometric information. In addition, true reflectance may be underestimated in shaded areas. In land cover classification, shaded areas are often left unclassified or simply assigned as a shadow class. Vegetation indices from remote sensing measurement are radiation-based measurements computed through spectral combination. They indicate vegetation properties and play an important role in remote sensing of forests. Airborne light detection and ranging (LiDAR) technology is an active remote sensing technique that produces a true orthophoto at a single wavelength. This study investigated three types of geometric lidar features where NDVI values fail to represent meaningful forest information. The three features include echo width, normalized eigenvalue, and standard deviation of the unit weight observation of the plane adjustment, and they can be derived from waveform data and discrete point clouds. Various feature combinations were carried out to evaluate the compensation of the three lidar features to vegetation detection in shaded areas. Echo width was found to outperform the other two features. Furthermore, surface characteristics estimated by echo width were similar to that by normalized eigenvalues. Compared to the combination of only NDVI and mean height difference, those including one of the three features had a positive effect on the detection of vegetation class.

SUMMARY While plasminogen activator inhibitor-1 (PAI-1) is known to potentiate cellular migration via proteolytic regulation, this adipokine is implicated as an oncogenic ligand in the tumor microenvironment. To understand the underlying paracrine mechanism, here, we conduct transcriptomic analysis of 1,898 endometrial epithelial cells (EECs) exposed and unexposed to PAI-1-secreting adipose stromal cells. The PAI-1-dependent action deregulates crosstalk among tumor-promoting and tumor-repressing pathways, including transforming growth factor β (TGF-β). When PAI-1 is tethered to lipoprotein receptor-related protein 1 (LRP1), the internalized signaling causes downregulation of SMAD4 at the transcriptional and post-translational levels that attenuates TGF-β-related transcription programs. Repression of genes encoding the junction and adhesion complex preferentially occurs in SMAD4-underexpressed EECs of persons with obesity. The findings highlight a role of PAI-1 signaling that renders ineffective intercellular communication for the development of adiposity-associated endometrial cancer., Graphical Abstract, In Brief Lin et al. demonstrate that PAI-1 secreted by adipose stromal cells interacts with LRP1 to repress TGF-β/SMAD4-regulated genes linked to cellular junction and adhesion complexes. This action disrupts cell-cell communication and facilitates the development of obesity-driven endometrial cancer.

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer (TAM) family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in NSCLC and its expression correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogated the effects of the AXL inhibitor, TP-0903, on EMT in NSCLC cells using transcriptomic and proteomic profiling. Methods: Atomic force microscopy, Western blot analysis, RNA sequencing, and mass cytometry (CyTOF) were used to evaluate the phenotypic, transcriptomic, and proteomic profiles of A549 cells treated with 40 nM TP-0903 or shAXL knockdown. A549 and H1650 NSCLC xenograft models were used to explore the consequences of AXL inhibition in vivo. Results: As expected, TP-0903 treatment attenuated AXL signaling and downstream phosphorylation in the A549 cells. Interestingly, the treatment also reduced gene expression responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1, and TAZ. Consistent with AXL inhibition, TP-0903 reversed the mesenchymal phenotype in A549 and H2009 cell lines and decreased their migratory potential in culture. The CyTOF analysis on TP-0903-treated cells identified resistant clones overexpressing TGFβ receptor II (TGFBR2) and TAZ proteins and displaying hybrid EMT phenotypes. TP-0903 was also active in suppressing A549 or H1650 tumor growth in vivo. Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 has excellent therapeutic promise in NSCLC and we speculate that TP-0903 can target mesenchymal transitional states in NSCLC, possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis. Citation Format: Josephine A. Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel A. Osmulski, Maria E. Gaczynska, Chiou-Miin Wang, Nicholas D. Lucio, Meizhen Chen, Chih-Wei Chou, Alia Nazarullah, Shellye R. Lampkin, Lianquin Qiu, David J. Bearss, Steve Warner, Lars Mouritsen, Mark Wade, Daniel DeArmond, Ruben Mesa, Nameer Kirma, Tim H.-M. Huang. AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B29.

Intercellular crosstalk between lung adenocarcinoma cells (LACs) and tumor-associated macrophages (TAMs) has been implicated in promoting cancer metastasis; however, how oncogenic signaling networks corroborate in this tumorigenic process remains unexplored. Our findings suggest that coordinated activation of AXL and JAK1-pSTAT3 signaling engenders a vicious cycle of dependence between LACs and TAMs for tumor progression. AXL-overexpressing LACs polarize TAMs to an M2-like phenotype in a JAK1-STAT3-dependent manner, facilitating the development of a proinflammatory tumor microenvironment. Conversely, M2-like TAMs release Gas6 ligand to enhance AXL-mediated cancer stemness and epithelial-to-mesenchymal transition (EMT) in LACs. Cytometry by time-of-flight (CyTOF) is a single-cell detection technology that allows for measurement of multiple protein markers in diverse cell subpopulations. We designed a 34-antibody CyTOF panel to identify immune cells, stromal cells, epithelial cells, oncogenic signaling, stemness and EMT components for LAC tumor profiling. CyTOF profiling of ~642,241 single cells detected 55 subpopulations among LAC cell lines (n=4 with co-culture conditions) and lung tumors (n=12), which were further differentiated based on their cell types (LACs, TAMs, stromal cells, granulocytes, natural killer, B and T lymphocytes). High AXL expression was noticed in tumor cell subpopulation while high pSTAT3 expression was detected in the TAM subpopulation. Moreover, pSTAT3 expression levels were increased in U937 differentiated macrophage after co-culture with A549 cells. This novel platform may facilitate the optimal selection of lung cancer patients for dual targeting of AXL and JAK1-pSTAT3.

Chromothripsis is an event of genomic instability leading to complex chromosomal alterations in cancer. Although studies have proposed models to explain its initiation, few discussed how transcription factors (TFs) influence chromothripsis for tumor progression. Frequent long-range chromatin interactions between TFs and targets may promote extensive translocations and copy-number alterations in proximal contact regions through inappropriate DNA stitching. Herein we surveyed genomic alterations of chromosome 17 in 1,014 breast tumors in the Cancer Genome Atlas (TCGA) cohort and found five adjoining regions from 17q11.1 to 17q24.1 being hotspots of chromothripsis. Inter-/intra-chromosomal rearrangements of these regions occurred more frequently in ERα-positive tumors than in ERα-negative tumors. In addition, the rearrangement sites were often mapped within or close to dense estrogen receptor 1 (ESR1) sites localized on the regions or other chromosomes. This chromothriptic event was linked to concordant upregulation of 96 loci that predominantly regulate cell-cycle machineries in advanced luminal tumors. Genome-editing analysis confirmed that an ESR1 transcription hub localized on 20q13 coordinately regulates a subset of these loci localized on 17q23 through long-range chromosome interactions. One of these loci, Tousled Like Kinase 2 (TLK2) known to participate in DNA damage checkpoint control, is an actionable target using phenothiazine antipsychotics (PTZs). The antiproliferative effect of PTZs was prominent in high TLK2-expressing cells, compared to low expressing cells. Collectively, we identified a group of luminal breast tumors displaying 17q-related chromothripsis for which antipsychotics can be repurposed as treatment adjuncts. Citation Format: Chun-Lin Lin. ESR1 related chromothripsis enhances concordant gene transcription on chromosome 17q11.1-q24.1 in luminal breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-05-17.

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro co-culture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n=141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients.

BACKGROUND Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci—SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. Prostate 75:1790–1801, 2015. © 2015 Wiley Periodicals, Inc.

Cyclophosphamide (CY) is a chemotherapeutic agent used for cancer and immunological diseases. It induces cytotoxicity of bone marrow and causes myelosuppression and extramedullary haematopoiesis (EMH) in treated patients. EMH is characterized with the emergence of multipotent haematopoietic progenitors most likely in the spleen and liver. Previous studies indicated that a Chinese medicine, ginsenoside Rg1, confers a significant effect to elevate the number of lineage (Lin(-) ) Sca-1(+) c-Kit(+) haematopoietic stem and progenitor cells (HSPCs) and restore the function of bone marrow in CY-treated myelosuppressed mice. However, whether the amelioration of bone marrow by Rg1 accompanies an alleviation of EMH in the spleen was still unknown. In our study, the cellularity and weight of the spleen were significantly reduced after Rg1 treatment in CY-treated mice. Moreover, the number of c-Kit(+) HSPCs was significantly decreased but not as a result of apoptosis, indicating that Rg1 alleviated EMH of the spleen induced by CY. Unexpectedly, the proliferation activity of c-Kit(+) HSPCs was only up-regulated in the spleen, but not in the bone marrow, after Rg1 treatment in CY-treated mice. We also found that a fraction of c-Kit(+) /CD45(+) HSPCs was simultaneously increased in the circulation after Rg1 treatment. Interestingly, the effects of Rg1 on the elevation of HSPCs in bone marrow and in the peripheral blood were suppressed in CY-treated splenectomized mice. These results demonstrated that Rg1 improves myelosuppression induced by CY through its action on the proliferation of HSPCs in EMH of the spleen and migration of HSPCs from the spleen to the bone marrow.

As advances in manufacture technology, leakage current increases dramatically in modern ICs. By turning off supply voltage in a low-power domain with power switches, power gating becomes a useful technique in resolving this problem. Since number and locations of power switches have great impact on chip area and IR-drop, an efficient and effective approach to insert power switches is required for the power gating designs. Unlike previous works using the greedy algorithm to handle this problem, this paper uses a simplified model to approximate required equivalent resistance of power switches in a low-power domain, and then determines number and types of power switches based on the value. In order to reduce impact on preplaced standard cells, we also propose a mathematical approach to find locations with less placement density to place power switches. The proposed methodology was integrated into a real-design flow. Experimental results demonstrate that our approach can insert less number of power switches and still satisfy the IR-drop constraint than other approaches.

Airborne light detection and ranging (LiDAR) technology is an active remote sensing technique that produces a true orthophoto at a single wavelength. LiDAR is not as dependent on the weather as optical sensors, and LiDAR features have been widely applied for characterizing land cover classes of interest. Instead of using point-based features for classification, object-based LiDAR features were employed in this study. In addition, instead of directly mining a data set with large numerical attributes, a preprocessing step, known as data discretization, was applied before data mining. The results show that although original data are simplified, mining results are concise, more meaningful and easy-to-interpret. In this way, the accuracy of land cover classification can be further improved.

Purpose: Little is known about adaptive selection of tumor cells transiting from in situ proliferation to distant colonization through blood circulation. This study used genomic, transcriptomic, and biophysical analyses at single-cell levels to explore biological and physical properties of circulating tumor cells (CTCs) undergoing hemodynamic stress. The aims are trying to understand how CTCs strive to survive in the bloodstream and to develop strategies for therapeutic intervention. Experimental Design: We compared genomic profiles of CTCs in blood and primary tumor cells shed in urine of prostate cancer patients to identify amplified regions preferentially retained in CTCs. Single-cell RNA-seq was used to confirm amplification-associated overexpression of genes in CTCs. Results: Among 261 recurrently amplified genomic regions in the analysis, 70 were found predominantly shown in CTCs relative to primary tumor cells. In line with the results at the genomic level, the transcriptomic data of CTCs demonstrate a great amount of cells showing high expression levels in the oxidative phosphorylation (OXPHOS) pathway compared to other hallmark gene sets. The finding suggests that tumor cells with these pre-existing genomic alterations were adaptively selected for transcription reprogramming during blood circulation. Specifically, amplified genes associated with OXPHOS were exploited by CTCs for alternative fuels. As to the upstream of this transcription event, we found the expression of MEN1, encoding menin known to form a transcription factor complex with the mixed-lineage leukemia protein (MLL) in prostate cancer cells, was positively correlated with 11 of the 14 OXPHOS loci in the Cancer Genomic Atlas prostate cancer cohort. In vitro assay showed that MEN1 knockdown by shRNA resulted in attenuation of both mRNA and protein expression of OXPHOS loci in PC-3 cells. Taken together pre-existing amplification of OXPHOS loci can be used as a transcription apparatus by menin for metabolic reprogramming of tumor cells in response to harsh microenvironments in the bloodstream. Conclusions: Single-cell profiling identified signaling pathways that are crucial for CTCs to survive in the bloodstream. The finding suggests that a metabolic shift from Warburg to OXPHOS metabolism can be associated with a hybrid mesenchymal-epithelial phenotype of CTCs. Moreover, the study demonstrates the feasibility of routinely conducting single-cell analysis of exfoliated tumor cells for minimally invasive monitoring of disease progression and treatment response in prostate cancer patients Citation Format: Chun-Lin Lin, Tan Xi, Chia-Nung Hung, Pawel A. Osmulski, Chih-Wei Chou, Meizhen Chen, Chiou-Miin Wang, Kohzoh Mitsuya, Nameer Kirma, Maria E. Gaczynska, Chun-Liang Chen, Tim H.-M. Huang. Amplification-associated upregulation of genes involved in oxidative phosphorylation for disseminated prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2766.

Estrogen stimulation triggers ERα binding to distant estrogen response elements (DEREs) through intra- or inter-chromatin interactions (Hsu et al. 2013; Hsu et al. 2015). Prolonged or repetitive stimulation causes alterations of histone marks and DNA methylation patterns in neighboring regions of DEREs, establishing three-dimensional structures for concordant transcription of estrogen-responsive genes. DEREs from multiple chromosome regions can be mobilized to form transcription depots for synchronized transcription of neighboring target genes. Heterochromatin protein 1 (HP1) acts as an anchor in regulating chromatin movement of DERE-mediated transcription assemblages in the nucleus (Hsu et al. 2015). Although HP1 is a fundamental unit of heterochromatin and acts a transcriptional repressor, we found that this protein may serve as a lamina buttress to support free movement of flexible chromatin in assembling transcription factories (Beisel and Paro 2011; Hsu et al. 2015). Neutralizing HP1 function deregulates the formation of DERE depots for concordant transcription of target genes. Furthermore, deletion of DEREs using the CRISPR/Cas9 genomic editing system alters transcription profiles of proliferation-associated networks, resulting in reduced cellular growth of luminal breast cancer cells. The epigenetic control of DERE-related chromatin movement has profoundly geographic influences on local genomic structures of gene clusters. Frequent chromatin interactions increase DNA breaks at or nearby looping sites, contributing to genomic instability in cancer cells (Kaiser and Semple 2018). Inappropriate DNA repair leads to genomic fusion and duplication of DEREs and target promoters. Amplified DEREs potentially cooperate with epigenetic machineries for long-range transcription regulation, leading to intensification of gene activation and repression for advanced development of luminal breast cancer. Specifically, we have identified DEREs on chromosome 20q13 frequently amplified and translocated to other chromosomes in this cancer subtype (Hsu et al. 2015). Genome-wide survey of The Cancer Genomic Atlas (TCGA) breast cancer cohort identified 17 contiguous chromosome regions that contain gene clusters (n=16 to 33 genes) with high correlation coefficients between their amplification and expression levels. Five of these identified regions were from chromosome 17q, and, moreover, upregulation of their gene expression was linked to luminal breast cancer with adverse clinical outcomes. Specifically, we found that six genes BCAS3, BRIP1, INTS2, MED13, METTL2A and TLK2 displayed concordantly high expression are located on the active or open topologically associating domains of 17q23 enriched with permissive chromatin marks H3K36me3 and H3K4me3. Complex translocation patterns between 17q23 and 20q13 were detected in breast tumors. The expression of these genes was increased in breast cancer cells after estrogen stimulation. However, this synchronized regulatory function was abolished in breast cancer cells harboring CRISPR/Cas9-knockout of 20q13 DEREs. These findings further establish that the 17q23 region can serve as an estrogen-driven transcriptional hub to promote ERα-mediated gene regulation and also extend previously published results showing that 17q23 genes are indeed DERE-bound ERα target genes. One of these DERE-regulated loci is Tousled Like Kinase 2 (TLK2), known to participate in checkpoint regulation and DNA damage response (Bruinsma et al. 2016), is an actionable target. Amplification-associated upregulation of TLK2 can be suppressed in breast cancer cells treated with phenothiazine antipsychotics, thioridazine, perphenazine and trifluoperazine (Sudeshna and Parimal 2010). Antiproliferative effects of phenothiazines were prominent especially in high TLK2-expressing cells, compared to those low TLK2-expressing cells that were relatively insensitive to phenothiazines. To further extend the potential use of TLK2 inhibition, we determined the efficacy of phenothiazine treatment on ex vivo cultures of patient-derived circulating tumor cells (CTCs). Enriched populations of CTCs were obtained from blood samples of breast cancer patients with metastatic disease. CTCs derived from patients with ERα-positive breast cancer appeared to be more sensitive to TLK2 inhibition compared to those of ERα-negative patients. Therefore, these antipsychotics can potentially be used as an adjunct for treating breast cancer tumors carrying DERE-mediated 17q23 amplification. Citations: Beisel C, Paro R. 2011. Silencing chromatin: comparing modes and mechanisms. Nature reviews Genetics 12: 123-135. Bruinsma W, van den Berg J, Aprelia M, Medema RH. 2016. Tousled-like kinase 2 regulates recovery from a DNA damage-induced G2 arrest. EMBO reports doi:10.15252/embr.201540767: e201540767. Hsu P-Y, Hsu H-K, Lan X, Juan L, Yan Pearlly S, Labanowska J, Heerema N, Hsiao T-H, Chiu Y-C, Chen Y et al. 2013. Amplification of Distant Estrogen Response Elements Deregulates Target Genes Associated with Tamoxifen Resistance in Breast Cancer. Cancer Cell 24: 197-212. Hsu PY, Hsu HK, Hsiao TH, Ye Z, Wang E, Profit AL, Jatoi I, Chen Y, Kirma NB, Jin VX et al. 2015. Spatiotemporal control of estrogen-responsive transcription in ERα-positive breast cancer cells. Oncogene 35: 2379. Kaiser VB, Semple CA. 2018. Chromatin loop anchors are associated with genome instability in cancer and recombination hotspots in the germline. Genome biology 19: 101-101. Sudeshna G, Parimal K. 2010. Multiple non-psychiatric effects of phenothiazines: A review. European Journal of Pharmacology 648: 6-14. Citation Format: Chun-Lin Lin, Tim Hui-Ming Huang. Long-range epigenetic control of estrogen-responsive transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr SY43-03.

Radiation therapy (RT) is a standard treatment for organ confined prostate cancer (PCa). Although dose escalation increases local control, further escalation hampers effectiveness due to toxicity. Additionally, intrinsic and acquired resistance to radiation causes progression to advanced metastatic PCa. Therefore, there is an urgency for addition of adjuvant therapies that synergize with radiation to improve therapeutic efficacy. We have identified a novel combination regimen (Nexrutine, Nx, a natural compound plus radiation) that demonstrated strong synergistic tumor growth inhibition in vitro (in multiple prostate cancer cell lines) and in transgenic adenocarcinoma of mouse prostate (TRAMP) model in part through downregulation of ribosomal protein S6K (RPS6KB1). To understand the precise molecular mechanism of synergy between Nx and radiation, we conducted transcriptome profiling of prostate cancer cells treated with single and combination modalities and followed up with pathway analysis, clinical validation, and in vitro gain and loss of function studies. Analyses of these data identified significant alterations in expression of genes across all experimental groups and genes that were unique to each group. Functional annotation using DAVID analysis of the top 200 genes in the combination group revealed ‘cell cycle regulation’ as the top pathway altered in the combination treatment. Remarkably, 22 genes enriched in the cell cycle pathway (including RPS6KB1) showed significant upregulation in high Gleason human prostate tumors. Interestingly cBioportal data indicated that alterations of RPS6KB1 in human prostate tumors is mutually exclusive with expression changes of AKT or NFκB. Computational molecular modeling studies showed that Berberine (Ber), one of the active constituents of Nx, interacts with RPS6KB1 in a novel mode. Additional biochemical and molecular investigations using RPS6KB1 KO cells divulged that KO cells are relatively (i) resistant to Ber and (ii) more sensitive to NFκB inhibitor-induced proliferation inhibition compared to wild type LNCaP cells. Collectively, our findings (i) identified RPS6KB1 as a direct target of Ber; and (ii) demonstrate that RPS6KB1 is a clinically relevant target of prostate cancer with possible role in radiosensitization. This work is supported in part by SABER*IRACDA grant K12GM11726 (ADC) and VA-Merit Award I01 BX 003876 (APK). Citation Format: Alison D. Clark, Suleman S. Hussain, Shih-Bo Huang, Roble G. Bedolla, Mohammad A. Obeidat, Niko Papanikolaou, Chun Lin Lin, Tim H. Huang, Yogesh Gupta, Yidong Chen, Hiroshi Miyamoto, Rita Ghosh, Addanki P. Kumar. Inhibition of ribosomal protein RPS6KB1 as a strategy to improve conventional therapy for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3943.

Dysregulated dynamics and trafficking of receptor tyrosine kinases (RTK) have been linked to oncogenesis and metastasis. The mechanisms for disfunction of RTK dynamics are emerging and still not clear. Using single-particle tracking (SPT) techniques, we studied the dynamics and trafficking patterns of epidermal growth factor receptor (EGFR) and underlying mechanisms using prostate cancer cells (PCa) (LNCaP, DU145 and PC3) exhibiting variant metastatic capability. Through SPT and super-resolution imaging, it was revealed that cortical actin disorganization modulates increased dynamics of EGFRs, including high EGFR diffusivity, enlarged EGFR confinement size on the plasma membrane and faster EGFR internalization in advanced invasive cells. The elevated EGFR dynamics were associated with advanced aggressive behaviors, and highly upregulated EGFR, EPHB and SRC signaling as identified using single cell RNA-seq. Strong EGFR activation with relatively low EGFR expression in PC3 implicated some other mechanisms, likely biophysical, beyond EGFR quantity. Moreover, the upregulated EPHB and SRC pathways have been shown to regulate actin organization and metastasis and our in silico analysis indicated that genes in the two pathways predict poor prognosis for disease free and survival statuses in The Cancer Genome Atlas prostate cancer patient cohort. To interrogate the roles of EPHB and SRC pathways in actin organization and EGFR dynamics, a series of drug inhibitions and siRNA knockdown were applied to PCa cells followed by mRNA profiling, evaluations of EGFR dynamics, and cell behaviors. Functional knockdown of EPHB2 and SRC led to decreased EGFR dynamics, cell proliferation, migration and invasion. The loss-of-function effects were more profound in advanced invasive PCa. In this study, we discovered the roles of EPHB and SRC pathways in modulating actin organization and EGFR dynamics and leading to aggressive metastatic phenotypes. Additionally, EGFR dynamics were potential biophysical parameters to differentiate the highly-invasive from the non- and less-invasive PCa. Thus, we believe that the SPT-based EGFR dynamics can serve as a new biophysical assay to probe the metastatic malignancy of cancer cells and to monitor their response to anti-cancer drug treatment. Citation Format: Yen-Liang Liu, Aaron M. Horning, Che-Kuang Lin, Brandon Lieberman, Chia-Nung Hung, Chih-Wei Chou, Chiou-Miin Wang, Michael A. Liss, Mirae Kim, Rohan Vasisht, Evan P. Perillo, Katherine Blocher, Hannah Horng, Xi Tian, Chun-Lin Lin, Andrew K. Dunn, Tim H.-M. Huang, Hsin-Chih Yeh, Chun-Liang Chen. Upregulated EPHB2 and SRC pathways modulate spatial EGFR dynamics and malignant phenotypes and predict poor prognosis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 173.

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in lung tumors, correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogate the effects of AXL inhibitor TP-0903 on EMT in lung adenocarcinoma cells using transcriptomic and proteomic profiling. Methods: Atomic force microscopy, Western blot analysis, RNA sequencing and mass cytometry (CyTOF) were all used to scrutinize the biomechanical properties, phenotypic, transcriptomic and proteomic profiles of A549 cells treated with 40nM TP0903 or shAXL knockdown. Results: TP-0903 attenuates total AXL/AXL phosphorylation and blunts transcriptional responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1 and TAZ. AXL knockdown or TP-0903 reverses EMT phenotype and reduces migration potential in A549 and H2009 adenocarcinoma cell lines. CyTOF data also identified resistant clones that overexpress TGFβ receptor II, TAZ protein and display hybrid EMT phenotypes. Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 study agent has excellent therapeutic promise in NSCLC and we speculate that TP-0903 drug can target epithelial to mesenchymal transitional states in lung cancer cells possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis. Citation Format: Josephine Amalia Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel Osmulski, Meizhen Chen, Chiou-Miin Wang, Nicholas L. L. Lucio, Nameer Kirma, Chih-Wei Chou, Maria E. Gaczynska, Alia Nazarullah, Mark Wade, Lars Mouritsen, Tim Huang. AXL inhibitor TP-0903 attenuates AXL-TGFbeta Hippo signaling axis in lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2193.

Endometrial cancer is the most common gynecologic cancer. It is divided into two major types, a less aggressive endometrioid type and a more aggressive serous type. Recent clinical observations underscore obesity as a major risk factor for endometrial cancer. In this study, we hypothesized that obesity can result in long term epigenetic modifications in the endometrial compartment, leading to the promotion of endometrial cancer. We examined the role of adipose-derived stem cells (ASCs), which are adipocyte progenitors, in the endometrial tumor microenvironment. By staining for ASCs in a tumor tissue microarray, we observed ASC infiltration that was specific to samples from obese patients, correlating with body mass index (R2=0.77, p Citation Format: Li-Ling Lin, Srikanth Polusani, Guangcun Huang, Chun-Lin Lin, Chiou-Miin Wang, Nicholas Lucio, Mikhail Kolonin, Alexes Daquinag, Pawel Osmulski, Bruce Nicholson, Edward Kost, Tim Huang, Nameer B. Kirma. Adipose-derived stem cell disruption of gap junction intercellular communication in obesity-associated endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5150.

// Guangcun Huang 1 , Pawel A. Osmulski 1 , Hakim Bouamar 2 , Devalingam Mahalingam 3 , Chun-Lin Lin 1 , Michael A. Liss 4 , Addanki Pratap Kumar 4, 5 , Chun-Liang Chen 1 , Ian M. Thompson 4 , Lu-Zhe Sun 2 , Maria E. Gaczynska 1 , Tim H.-M. Huang 1 1 Departments of Molecular Medicine Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 2 Departments of Cellular and Structural Biology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 3 Departments of Medicine Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 4 Departments of Urology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 5 Departments of Radiation Oncology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Correspondence to: Tim H.-M. Huang, email: huangt3@uthscsa.edu Maria E. Gaczynska, email: gaczynska@uthscsa.edu Keywords: tumor metastasis, circulating tumor cells, epithelial-mesenchymal transition (EMT), transforming growth factor-β (TGF-β), positive feedback signaling Received: August 08, 2016 Accepted: October 12, 2016 Published: October 21, 2016 ABSTRACT Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII -edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro . Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Background The Pap smear has remained the foundation for cervical cancer screening for over 70 years. With advancements in molecular diagnostics, primary high-risk human papillomavirus (hrHPV) screening has recently become an accepted stand-alone or co-test with conventional cytology. However, both diagnostic tests have distinct limitations. The aim of this study was to determine the association between HPV genotypes and cellular epigenetic modifications in three grades of cervical cytology for screening biomarker discovery. Methods This prospective, cross-sectional study used residual liquid-based cytology samples for HPV genotyping and epigenetic analysis. Extracted DNA was subjected to parallel polymerase chain reactions using three primer sets (MY09/11, FAP59/64, E6-E7 F/B) for HPV DNA amplification. HPV+ samples were genotyped by DNA sequencing. Promoter methylation of four candidate tumor suppressor genes (adenylate cyclase 8 (ADCY8), cadherin 8, type 2 (CDH8), MGMT, and zinc finger protein 582 (ZNF582)) out of 48 genes screened was quantified by bisulfite-pyrosequencing of genomic DNA. Independent validation of methylation profiles was performed by analyzing data from cervical cancer cell lines and clinical samples from The Cancer Genome Atlas (TCGA). Results Two hundred seventy-seven quality cytology samples were analyzed. HPV was detected in 31/100 (31 %) negative for intraepithelial lesion or malignancy (NILM), 95/100 (95 %) low-grade squamous intraepithelial lesion (LSIL), and 71/77 (92 %) high-grade squamous intraepithelial lesion (HSIL) samples. The proportion of IARC-defined carcinogenic HPV types in sequenced samples correlated with worsening grade: NILM 7/29 (24 %), LSIL 53/92 (58 %), and HSIL 65/70 (93 %). Promoter methylation of ADCY8, CDH8, and ZNF582 was measured in 170 samples: NILM (N = 33), LSIL (N = 70), and HSIL (N = 67) also correlated with worsening grade. Similar hypermethylation patterns were found in cancer cell lines and TCGA samples. The combination of four biomarkers, i.e., HPV genotype and three-gene promoter methylation, predicted HSIL (AUC 0.89) better than HPV alone (AUC 0.74) by logistic regression and probabilistic modeling. Conclusions HPV genotype and DNA methylation of ADCY8, CDH8, and ZNF582 are correlated with cytological grade. Collectively, these biomarkers may serve as a molecular classifier of Pap smears. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0263-9) contains supplementary material, which is available to authorized users.

With the invention of Microsoft Kinect sensor, human-computer interaction is gaining its attention and becoming available for widespread use. The previous study presented a method of Kinect-based mid-air handwritten digit recognition for Android smart phones with a recognition accuracy of only about 94.6%. In this paper, we propose an improved method based on the normalizing and scaling of path order coordinates. With that, the proposed method leads to accuracy elevation and executing time reduction. Experimental results show an average recognition accuracy rate of 96.8% was achieved for each number.