Search

Background: Radiotherapy is a standard treatment option for early glottic carcinoma (stage I and II) with a fraction size of 2-2.2 Gy over 5-7 weeks. This study evaluates the outcome and prognostic factors of a 3-week hypofractionated treatment in early glottic malignancy., Materials and Methods: The case records of 329 eligible patients with stage I and II glottic carcinoma recorded at the institution from 2003 to 2008 were retrospectively analysed. All patients were treated in a Cobalt-60 machine to a dose of 52.5 Gy in 15 fractions (3.5 Gy/fraction) over 3 weeks., Results: Eighty-three percent had stage I disease. The local control rate at 5 years was 91.9%. On univariate analysis, stage I and II patients without subglottic extension had better local control. Disease extension to the subglottis fared poorly on multivariate analysis. After salvage treatment, the 5-year disease-free survival rate was 96.1% and the functional larynx preservation rate was 94.9% for stage I and 83.9% for stage II. The rate of severe complications was 2.1%., Conclusion: Comparable results with low morbidity are achievable with a 3-week hypofractionation in early glottic cancers and it offers better patient convenience., Highlights: In early glottic cancer, hypofractionated radiation provides excellent local control.Subglottic extension is a poor prognostic factor.5-year disease-free survival rate of 96.1%.5-year functional larynx preservation rate of 94.9%.Severe complication rate of 2.1%., Competing Interests: None of the authors have any conflict of interest in the publication of this manuscript., (© the authors; licensee ecancermedicalscience.)

Preclinical studies of primary cancer cells are typically done after tumors are removed from patients or animals at ambient atmospheric oxygen (O 2 , ~21%). However, O 2 concentrations in organs are in the ~3 to 10% range, with most tumors in a hypoxic or 1 to 2% O 2 environment in vivo. Although effects of O 2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anticancer agents is routinely examined at ambient O 2 . Here, we demonstrate that tumors collected, processed, and propagated at physiologic O 2 compared to ambient air display distinct differences in key signaling networks including LGR5/WNT, YAP, and NRF2/KEAP1, nuclear reactive oxygen species, alternative splicing, and sensitivity to targeted therapies. Therefore, evaluating cancer cells under physioxia could more closely recapitulate their physiopathologic status in the in vivo microenvironment.

Cancer cells develop tactics to circumvent the interventions by desensitizing themselves to interventions. Amongst many, the principle routes of desensitization include a) activation of survival pathways (e.g. NF-kB, PARP) and b) downregulation of cell death pathways (e.g. CD95/CD95L). As a result, it requires high therapeutic dose to achieve tumor regression which, in turn damages normal cells through the collateral effects. Methods are needed to sensitize the low and non-responsive resistant tumor cells including cancer stem cells (CSCs) in order to evoke a better response from the current treatments. Current treatments including chemotherapy can induce cell death only in bulk cancer cells sparing CSCs and cancer resistant cells (CRCs) which are shown to be responsible for high recurrence of disease and low patient survival. Here, we report several novel tumor targeted sensitizers derived from the natural Vitamin E analogue (AMP-001-003). The drug design is based on a novel concept "A priori activation of apoptosis pathways of tumor technology (AAAPT) which is designed to activate specific cell death pathways and inhibit survival pathways simultaneously and selectively in cancer cells sparing normal cells. Our results indicate that AMP-001-003 sensitize various types of cancer cells including MDA-MB-231 (triple negative breast cancer), PC3 (prostate cancer) and A543 (lung cancer) cells resulting in reducing the IC-50 of doxorubicin in vitro when used as a combination. At higher doses, AMP-001 acts as an anti-tumor agent on its own. The synergy between AMP-001 and doxorubicin could pave a new pathway to use AAAPT leading molecules as neoadjuvant to chemotherapy to achieve better efficacy and reduced off-target toxicity compared to the current treatments., Competing Interests: The corresponding author, on behalf of all authors declares no competing financial or non-financial interest. Affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Sarcopenia is characterised by progressive and extensive skeletal muscle degeneration and is associated with functional decline. Sarcopenia has primary and secondary aetiology, arising as a result of the ageing process or through chronic cytokine-mediated inflammation (associated with health conditions including cancer), respectively. Diagnosis of sarcopenia is dependent upon detection of reduced skeletal muscle strength, mass and performance. A combination of non-radiological and radiological methods can be used to assess each of these in turn to accurately diagnose sarcopenia. Sarcopenia is known to adversely affect outcomes of patients with various forms of cancer. Early identification of sarcopenia is imperative in improving patient care and overall prognosis. Various interventions, such as resistance exercise, nutritional support, and amino acid and vitamin supplementation have shown promise in the management of sarcopenia. However, further insight into novel interventions and indeed, assessment of the benefits of management of sarcopenia in terms of survival, are required to better support cancer patients., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Background: Predicting outcomes and response to therapy through biomarkers is a major challenge in cancer research. In previous studies, we suggested that inappropriate "normal" tissue samples used for comparison with tumors, inter-individual heterogeneity in gene expression, and genetic ancestry all influence biomarker expression in tumors. The aim of this study was to investigate these factors in breast cancer using breast tissues from healthy women and normal tissue adjacent to tumor (NAT) with matrix metalloproteinase 7 (MMP7) as a candidate biomarker., Study Design: RNA sequencing was performed on primary luminal progenitor cells from healthy breast, NATs, and tumors to identify transcriptomes enriched in NATs and breast cancer. Expression of select genes was validated via quantitative reverse transcription polymerase chain reaction of RNA and via immunohistochemistry of a tissue microarray of normal, NAT, and tumor samples of different genetic ancestry., Results: Twenty-six genes were significantly overexpressed in NATs and tumors compared with healthy controls at messenger RNA level and formed a para-inflammatory network. MMP7 had the greatest expression in tumor cells, with upregulation confirmed by quantitative reverse transcription polymerase chain reaction. Tumor-enriched but not NAT-enriched expression of MMP7 compared with healthy controls was reproduced at protein levels. When stratified by genetic ancestry, tumor-specific increase of MMP7 reached statistical significance in women of European ancestry., Conclusions: Transcriptome differences across healthy, NAT, and tumor tissue in breast cancer demonstrate an active para-inflammatory network in NATs and indicate unsuitability of NATs as "normal controls" in biomarker discovery. The discordance between transcriptomic and proteomic MMP7 expression in NATs and the influence of genetic ancestry on its protein expression highlight the complexity in developing universally acceptable biomarkers of breast cancer and the importance of genetic ancestry in biomarker development., (Copyright © 2020 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Until recently, men with metastatic prostate cancer were commenced on androgen deprivation therapy at diagnosis, followed by sequential lines of treatment with the development of castration resistance. However, the results of recent clinical trials, which revealed that the addition of radiotherapy to the prostate to a dose of 55 to 60 Gy in 20 fractions over 4 weeks in patients with low-volume metastatic hormone-sensitive prostate cancer, in addition to androgen deprivation therapy and another systemic treatment option, either docetaxel, abiraterone, enzalutamide, or apalutamide, has led to a paradigm change in the management of this disease.

Functional modeling of normal breast epithelial hierarchy and stromal-epithelial cell interactions have been difficult due to inability to obtain sufficient stem-progenitor-mature epithelial and stromal cells. Recently reported epithelial reprogramming assay has partially overcome this limitation, but cross-contamination of cells from the feeder layer is a concern. The purpose of this study was to develop a feeder-layer-independent and inexpensive method to propagate multiple cell types from limited tissue resources. Cells obtained after enzymatic digestion of tissues collected at surgery or by core-needle biopsies were plated on tissue culture dishes precoated with laminin-5-rich-conditioned media from the rat bladder tumor cell line 804G and a defined growth media with inhibitors of ROCK, TGFβ, and BMP signaling. Cells were characterized by flow cytometry, mammosphere assay, 3D cultures, and xenograft studies. Cells from the healthy breasts included CD10 + /EpCAM - basal/myoepithelial, CD49f + /EpCAM + luminal progenitor, CD49f - /EpCAM + mature luminal, CD73 + /EpCAM + /CD90 - rare endogenous pluripotent somatic stem, CD73 + /CD90 + /EpCAM - , estrogen receptor alpha-expressing ALCAM (CD166) + /EpCAM + , and ALDFLUOR + stem/luminal progenitor subpopulations. Epithelial cells were luminal (KRT19 + ), basal (KRT14 + ), or dual-positive luminal/basal hybrid cells. While breast cells derived from BRCA1, BRCA2, and PALB2 mutation carriers did not display unique characteristics, cells from women with breast cancer-protective alleles showed enhanced differentiation. Cells could also be propagated from primary tumors and metastasis of breast, ovarian, and pancreatic cancer-neuroendocrine subtype. Xenograft studies confirmed tumorigenic properties of tumor-derived cells. IMPLICATIONS: Our method expands the scope of individualized studies of patient-derived cells and provides resources to model epithelial-stromal interactions under normal and pathologic conditions., (©2019 American Association for Cancer Research.)

Cell-type origin is one of the factors that determine molecular features of tumors, but resources to validate this concept are scarce because of technical difficulties in propagating major cell types of adult organs. Previous attempts to generate such resources to study breast cancer have yielded predominantly basal-type cell lines. We have created a panel of immortalized cell lines from core breast biopsies of ancestry-mapped healthy women that form ductal structures similar to normal breast in 3D cultures and expressed markers of major cell types, including the luminal-differentiated cell-enriched ERα-FOXA1-GATA3 transcription factor network. We have also created cell lines from PROCR (CD201) + /EpCAM - cells that are likely the "normal" counterpart of the claudin-low subtype of breast cancers. RNA-seq and PAM50-intrinsic subtype clustering identified these cell lines as the "normal" counterparts of luminal A, basal, and normal-like subtypes and validated via immunostaining with basal-enriched KRT14 and luminal-enriched KRT19. We further characterized these cell lines by flow cytometry for distribution patterns of stem/basal, luminal-progenitor, mature/differentiated, multipotent PROCR + cells, and organogenesis-enriched epithelial/mesenchymal hybrid cells using CD44/CD24, CD49f/EpCAM, CD271/EpCAM, CD201/EpCAM, and ALDEFLUOR assays and E-cadherin/vimentin double staining. These cell lines showed interindividual heterogeneity in stemness/differentiation capabilities and baseline activity of signaling molecules such as NF-κB, AKT2, pERK, and BRD4. These resources can be used to test the emerging concept that genetic variations in regulatory regions contribute to widespread differences in gene expression in "normal" conditions among the general population and can delineate the impact of cell-type origin on tumor progression. Significance: In addition to providing a valuable resource for the breast cancer research community to investigate cell-type origin of different subtypes of breast cancer, this study highlights interindividual differences in normal breast, emphasizing the need to use "normal" cells from multiple sources as controls to decipher the effects of cancer-specific genomic aberrations. Cancer Res; 78(17); 5107-23. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: The majority of estrogen receptor-positive (ERα + ) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown., Methods: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα + MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses., Results: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14 + /KRT19 + and CD49f + /EpCAM + phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα + /PR + /HER2 - tumors, was associated with poor outcome., Conclusion: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα + breast cancers.

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

Breast cancer progression is associated with systemic effects, including functional limitations and sarcopenia without the appearance of overt cachexia. Autocrine/paracrine actions of cytokines/chemokines produced by cancer cells mediate cancer progression and functional limitations. The cytokine-inducible transcription factor NF-κB could be central to this process, as it displays oncogenic functions and is integral to the Pax7:MyoD:Pgc-1β:miR-486 myogenesis axis. We tested this possibility using the MMTV-PyMT transgenic mammary tumor model and the NF-κB inhibitor dimethylaminoparthenolide (DMAPT). We observed deteriorating physical and functional conditions in PyMT + mice with disease progression. Compared with wild-type mice, tumor-bearing PyMT + mice showed decreased fat mass, impaired rotarod performance, and reduced grip strength as well as increased extracellular matrix (ECM) deposition in muscle. Contrary to acute cachexia models described in the literature, mammary tumor progression was associated with reduction in skeletal muscle stem/satellite-specific transcription factor Pax7. Additionally, we observed tumor-induced reduction in Pgc-1β in muscle, which controls mitochondrial biogenesis. DMAPT treatment starting at 6 to 8 weeks age prior to mammary tumor occurrence delayed mammary tumor onset and tumor growth rates without affecting metastasis. DMAPT overcame cancer-induced functional limitations and improved survival, which was accompanied with restoration of Pax7, Pgc-1β, and mitochondria levels and reduced ECM levels in skeletal muscles. In addition, DMAPT restored circulating levels of 6 out of 13 cancer-associated cytokines/chemokines changes to levels seen in healthy animals. These results reveal a pharmacological approach for overcoming cancer-induced functional limitations, and the above-noted cancer/drug-induced changes in muscle gene expression could be utilized as biomarkers of functional limitations. Mol Cancer Ther; 16(12); 2747-58. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Aim: To determine the systematic error ( ∑ ), random error ( σ ) and derive PTV margin at different levels of the target volumes in Nasopharyngeal Cancer (NPC)., Materials and Methods: A retrospective offline review was done for patients who underwent IMRT for NPC from June 2015 to May 2016 at our institution. Alternate day kV images were matched with digitally reconstructed radiographs to know the setup errors. All radiographs were matched at three levels - the clivus, third cervical (C3) and sixth cervical (C6) vertebra. The shifts in positions along the vertical, longitudinal and lateral axes were noted and the ∑ and σ at three levels were calculated. PTV margins were derived using van Herk's formula., Results: Twenty patients and 300 pairs of orthogonal portal films were reviewed. The ∑ for the clivus, C3 and C6 along vertical, longitudinal and lateral directions were 1.6 vs. 1.8 vs. 2 mm; 1.2 vs. 1.4 vs. 1.4 mm and 0.9 vs. 1.6 and 2.3 mm, respectively. Similarly, the random errors were 1.1 vs. 1.4 vs. 1.8 mm; 1.1 vs. 1.2 vs. 1.2 mm and 1.2 vs. 1.3 vs. 1.6 mm. The PTV margin at the clivus was 4.4 mm along the vertical, 4 mm along the longitudinal direction and 3.2 m in the lateral direction. At the C3 level, it was 5.5 mm in the vertical, 5 mm in the lateral direction and 4.4 mm in the longitudinal direction. At the C6 level, it was 6.4 mm in the vertical, 6.9 mm in the lateral direction and 4.4 mm in the longitudinal direction., Conclusion: A differential margin along different levels of target may be necessary to adequately cover the target.

Aim: To determine the efficacy of belly board device in patients receiving postoperative radiation for gynecological malignancies in terms of setup error and acute small bowel toxicity., Materials and Methods: Patients requiring postoperative radiation for gynecological malignancies were prospectively randomized to either treatment with supine position (supine arm) or prone position using belly board (prone arm). Each patient underwent computed tomography simulation in the assigned treatment position, and a three-dimensional conformal radiation was planned. Weekly two to three treatment sessions were verified using portal imaging and setup errors were noted. All patients were reviewed weekly to assess for symptoms and toxicity using a structured format. The systematic and random errors were calculated along the three axes., Results: Twenty-four patients were randomized and 22 patients were available for the final analysis. The systematic error in supine arm versus prone arm was 3.9 versus 3.5 mm, 2.1 versus 4.8 mm and 3.1 versus 3.1 mm along lateral, antero-posterior (AP) and cranio-caudal (CC) direction. The random error in supine arm versus prone arm was 5 versus 3.9 mm, 2.9 mm versus 4.4 mm and 4.3 versus 3.4 mm along lateral, AP and CC direction. The calculated planning target volume margin for supine arm was 1.3, 0.7, and 1.0 cm and margin for prone arm was 1.1, 1.5, and 1.0 cm along lateral, AP, and CC direction, respectively. One patient in supine arm developed Grade 3 toxicity., Conclusion: The systematic error and random error is more along AP direction for prone position. The acute small bowel toxicity was less using belly board.

There is a need to individualize assays for tumor molecular phenotyping, given variations in the differentiation status of tumor and normal tissues in different patients. To address this, we performed single-cell genomics of breast tumors and adjacent normal cells propagated for a short duration under growth conditions that enable epithelial reprogramming. Cells analyzed were either unselected for a specific subpopulation or phenotypically defined as undifferentiated and highly clonogenic ALDH + /CD49f + /EpCAM + luminal progenitors, which express both basal cell and luminal cell-enriched genes. We analyzed 420 tumor cells and 284 adjacent normal cells for expression of 93 genes that included a PAM50-intrinsic subtype classifier and stemness-related genes. ALDH + /CD49f + /EpCAM + tumor and normal cells clustered differently compared with unselected tumor and normal cells. PAM50 gene-set analyses of ALDH + /CD49f + /EpCAM + populations efficiently identified major and minor clones of tumor cells, with the major clone resembling clinical parameters of the tumor. Similarly, a stemness-associated gene set identified clones with divergent stemness pathway activation within the same tumor. This refined expression profiling technique distinguished genes truly deregulated in cancer from genes that identify cellular precursors of tumors. Collectively, the assays presented here enable more precise identification of cancer-deregulated genes, allow for early identification of therapeutically targetable tumor cell subpopulations, and ultimately provide a refinement of precision therapeutics for cancer treatment. Cancer Res; 77(10); 2759-69. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Aim: This study aims to identify clinical features, treatment outcomes, and prognostic factors for relapse and survival in patients with testicular seminoma., Materials and Methods: Retrospective analysis of all patients with pure seminoma treated at our center during over a decade (January 2005-December 2014) was carried out. Patient demographics, tumor characteristics, and treatment details and pattern of recurrence were recorded in a structured format, and disease-free survival and overall survival were calculated., Results: Sixty-three patients' case records were included in the analysis. Ten patients developed disease in the undescended testis. All patients underwent orchiectomy as the initial treatment procedure. Majority of the patients were Stage I (57.14%) followed by Stage II (39.6%). Among the patients with Stage I, 55.5% received adjuvant chemotherapy while 22.2% received adjuvant radiation and the rest opted for surveillance. The compliance for active surveillance was very poor. Among patients with Stage II disease, majority (80%) were treated with adjuvant chemotherapy and the rest with radiation. At a median follow-up of 49 months, there were four recurrences of which three were salvaged successfully, and one patient remained alive with disease. There were no disease-related deaths., Conclusions: Testicular seminoma remains to be relatively low and majority of them presented with Stage I disease and single agent carboplatin appeared to be the preferred adjuvant treatment. Advanced disease patients were treated with etoposide and cisplatin/bleomycin, etoposide and cisplatin chemotherapy and the clinical outcome is comparable with the Western literature.

Unlabelled: Adipose-derived stem cells (ASC) have received considerable attention in oncology because of the known direct link between obesity and cancer as well as the use of ASCs in reconstructive surgery after tumor ablation. Previous studies have documented how cancer cells commandeer ASCs to support their survival by altering extracellular matrix composition and stiffness, migration, and metastasis. This study focused on delineating the effects of ASCs and adipocytes on the self-renewal of stem/progenitor cells and hierarchy of breast epithelial cells. The immortalized breast epithelial cell line MCF10A, ductal carcinoma in situ (DCIS) cell lines MCF10DCIS.com and SUM225, and MCF10A-overexpressing SRC oncogene were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profiles. Interestingly, ASCs promoted the self-renewal of all cell types except SUM225. ASC coculture or treatment with ASC conditioned media altered the number of CD49f(high)/EpCAM(low) basal/stem-like and CD49f(medium)/EpCAM(medium) luminal progenitor cells. Among multiple factors secreted by ASCs, IFNγ and hepatocyte growth factor (HGF) displayed unique actions on epithelial cell hierarchy. IFNγ increased stem/progenitor-like cells while simultaneously reducing the size of mammospheres, whereas HGF increased the size of mammospheres with an accompanying increase in luminal progenitor cells. ASCs expressed higher levels of HGF, whereas adipocytes expressed higher levels of IFNγ. As luminal progenitor cells are believed to be prone for transformation, IFNγ and HGF expression status of ASCs may influence susceptibility for developing breast cancer as well as on outcomes of autologous fat transplantation on residual/dormant tumor cells., Implications: This study suggests that the ratio of ASCs to adipocytes influences cancer cell hierarchy, which may impact incidence and progression. Mol Cancer Res; 14(7); 660-71. ©2016 AACR., (©2016 American Association for Cancer Research.)

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations.

The transcription factor nuclear factor-kappaB (NF-κB) is constitutively active in several cancers and is a target of therapeutic development. We recently developed dimethylaminoparthenolide (DMAPT), a clinical grade water-soluble analog of parthenolide, as a potent inhibitor of NF-κB and demonstrated in vitro and in vivo anti-tumor activities in multiple cancers. In this study, we show DMAPT is an epigenetic modulator functioning in an NF-κB-dependent and -independent manner. DMAPT-mediated NF-κB inhibition resulted in elevated histone H3K36 trimethylation (H3K36me3), which could be recapitulated through genetic ablation of the p65 subunit of NF-κB or inhibitor-of-kappaB alpha super-repressor overexpression. DMAPT treatment and p65 ablation increased the levels of H3K36 trimethylases NSD1 (KMT3B) and SETD2 (KMT3A), suggesting that NF-κB directly represses their expression and that lower H3K36me3 is an epigenetic marker of constitutive NF-κB activity. Overexpression of a constitutively active p65 subunit of NF-κB reduced NSD1 and H3K36me3 levels. NSD1 is essential for DMAPT-induced expression of pro-apoptotic BIM, indicating a functional link between epigenetic modification and gene expression. Interestingly, we observed enhanced H4K20 trimethylation and induction of H4K20 trimethylase KMT5C in DMAPT-treated cells independent of NF-κB inhibition. These results add KMT5C to the list NF-κB-independent epigenetic targets of parthenolide, which include previously described histone deacetylase 1 (HDAC-1) and DNA methyltransferase 1. As NSD1 and SETD2 are known tumor suppressors and loss of H4K20 trimethylation is an early event in cancer progression, which contributes to genomic instability, we propose DMAPT as a potent pharmacologic agent that can reverse NF-κB-dependent and -independent cancer-specific epigenetic abnormalities.

Circulating microRNAs (miRNA) are emerging as important biomarkers of various diseases, including cancer. Intriguingly, circulating levels of several miRNAs are lower in patients with cancer compared with healthy individuals. In this study, we tested the hypothesis that a circulating miRNA might serve as a surrogate of the effects of cancer on miRNA expression or release in distant organs. Here we report that circulating levels of the muscle-enriched miR486 is lower in patients with breast cancer compared with healthy individuals and that this difference is replicated faithfully in MMTV-PyMT and MMTV-Her2 transgenic mouse models of breast cancer. In tumor-bearing mice, levels of miR486 were relatively reduced in muscle, where there was elevated expression of the miR486 target genes PTEN and FOXO1A and dampened signaling through the PI3K/AKT pathway. Skeletal muscle expressed lower levels of the transcription factor MyoD, which controls miR486 expression. Conditioned media (CM) obtained from MMTV-PyMT and MMTV-Her2/Neu tumor cells cultured in vitro were sufficient to elicit reduced levels of miR486 and increased PTEN and FOXO1A expression in C2C12 murine myoblasts. Cytokine analysis implicated tumor necrosis factor α (TNFα) and four additional cytokines as mediators of miR486 expression in CM-treated cells. Because miR486 is a potent modulator of PI3K/AKT signaling and the muscle-enriched transcription factor network in cardiac/skeletal muscle, our findings implicated TNFα-dependent miRNA circuitry in muscle differentiation and survival pathways in cancer., (©2014 American Association for Cancer Research.)

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating immunoenrichment prior to rapid microwave and magnetic (IM(2) ) sample preparation, might enable correlation of the relative expression of CD47 and other low abundance proteins to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, anti-CD47 antibodies were used to enrich for low abundance CD47 prior to microwave and magnetic proteomics in EAE. Decoding protein expression at each time point, with CD47-immunoenriched samples and targeted proteomic analysis, enabled peptides from the low abundance proteins to be precisely quantified throughout disease progression, including: CD47: 86-99, corresponding to the "marker of self" overexpressed by myelin that prevents phagocytosis, or "cellular devouring," by microglia and macrophages; myelin basic protein: 223-228, corresponding to myelin basic protein; and migration inhibitory factor: 79-87, corresponding to a proinflammatory cytokine that inhibits macrophage migration. While validation in a larger cohort is underway, we conclude that IM(2) proteomics is a rapid method to precisely quantify peptides from CD47 and other low abundance proteins throughout disease progression in EAE. This is likely due to improvements in selectivity and sensitivity, necessary to partially overcome masking of low abundance proteins by high abundance proteins and improve dynamic range., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We hypothesized that quantitative MS/MS-based proteomics at multiple time points, incorporating rapid microwave and magnetic (M(2) ) sample preparation, could enable relative protein expression to be correlated to disease progression in the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis. To test our hypothesis, microwave-assisted reduction/alkylation/digestion of proteins from brain tissue lysates bound to C8 magnetic beads and microwave-assisted isobaric chemical labeling were performed of released peptides, in 90 s prior to unbiased proteomic analysis. Disease progression in EAE was assessed by scoring clinical EAE disease severity and confirmed by histopathologic evaluation for central nervous system inflammation. Decoding the expression of 283 top-ranked proteins (p <0.05) at each time point relative to their expression at the peak of disease, from a total of 1191 proteins observed in four technical replicates, revealed a strong statistical correlation to EAE disease score, particularly for the following four proteins that closely mirror disease progression: 14-3-3ε (p = 3.4E-6); GPI (p = 2.1E-5); PLP1 (p = 8.0E-4); PRX1 (p = 1.7E-4). These results were confirmed by Western blotting, signaling pathway analysis, and hierarchical clustering of EAE risk groups. While validation in a larger cohort is underway, we conclude that M(2) proteomics is a rapid method to quantify putative prognostic/predictive protein biomarkers and therapeutic targets of disease progression in the EAE animal model of multiple sclerosis., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two prototype 24-unit microbioreactors are presented and reviewed for their relative merits. The first used a standard 24-well plate as the template, while the second consisted of 24-discrete units. Both systems used non-invasive optical sensors to monitor pH and dissolved oxygen. The systems were used to cultivate Escherichia coli. Both designs had their merits and the results obtained are presented. In addition, dissolved oxygen control was demonstrated at the milliliter scale and 24 simultaneously monitored fermentations were successfully carried out. These results demonstrated high quality high throughput bioprocessing and provide important insights into operational parameters at small scale.

We present a new computer-controlled, electromechanical system for performing simultaneous extension, inflation, and torsion experiments on cylindrical segments of natural and artificial blood vessels. Specimens are tested while immersed in a temperature-controlled, oxygenated, physiologic solution. Deformations are measured within a central region of the specimen using noncontacting video methods. The associated axial loads, luminal pressures, and torques are measured with standard transducers. Data are collected and stored on-line, and are used in the feedback control of experimental protocols, which are prescribed using custom interactive software. Finally, we present illustrative data obtained from canine aortas and common carotid arteries.