Ali Khammanivong | University of Minnesota - Twin Cities (original) (raw)

Papers by Ali Khammanivong

Research paper thumbnail of Content Alerts

This article cites 38 articles, 19 of which can be accessed free

Research paper thumbnail of Molecular Cancer

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in

Research paper thumbnail of Abstract 671: Development of an exosomal biomarker signature to detect minimal residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning

Clinical Research (Excluding Clinical Trials)

Research paper thumbnail of Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux

Frontiers in Oncology

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few... more Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol...

Research paper thumbnail of Abstract B22: A method for early detection of hemangiosarcoma in dogs

Poster Presentations - Proffered Abstracts

Research paper thumbnail of Abstract B59: Validation of an exosomal osteosarcoma-associated gene signature in dogs with osteosarcoma

Poster Presentations - Proffered Abstracts

Research paper thumbnail of Abstract 1845: Propranolol reduces hemangiosarcoma cell viability by disrupting lipid homeostatic pathways

Experimental and Molecular Therapeutics

Research paper thumbnail of Abstract 4592: Early detection for strategic prevention of a terminal canine cancer: A model to reduce the impact of cancer in our society

Molecular and Cellular Biology / Genetics

Cancer mortality has surpassed all other causes of human death in parts of the developed world. D... more Cancer mortality has surpassed all other causes of human death in parts of the developed world. Domestic dogs and cats are the only other species where cancer has an equivalent impact in overall mortality. Rapid gains in longevity over the past two centuries are sufficient to explain the increased incidence of cancer in all three species. Not only does longevity increase the probability of exposures to environmental mutagens, but it also allows for introduction of mutations in each somatic cell replication. In this context, an increase in the incidence of cancer becomes almost inevitable because the evolutionary, cancer protective adaptations of humans, dogs, and cats are insufficient to provide protection over the 2 to 6-fold extension in lifespan achieved by these species. We believe this problem is addressable by developing tests to detect malignancies at their earliest stages and pairing these tests with strategic interventions to eliminate tumors before they form. To test this premise, we have developed a blood test to detect the presence of hemangiosarcoma in dogs, a rapidly fatal malignancy. The shorter lifespan of dogs, compared to humans, and the similar shorter latency of disease provide an ideal opportunity to assess the feasibility of our approach. The test uses flow cytometry for detection of rare, non-leukocyte nucleated cells in blood, combining 42 parameters for analysis in a machine learning environment. Samples from 126 dogs with a confirmed diagnosis of hemangiosarcoma (n=28); other cancers (n=29); benign vascular pathology of the spleen (n=27); or apparently healthy and under 4 years of age (n=41) were used for training and 10-fold cross validation. The expected sensitivity and specificity for the diagnosis of hemangiosarcoma in this training set were approximately 90% and 95%, respectively. We then used our trained models to assign a risk category to apparently healthy dogs, 6 years of age or older, from three breeds with a predisposition for hemangiosarcoma (up to 20% lifetime risk): samples from 105 golden retrievers, 52 boxers and 52 Portuguese Water Dogs (N=209) were included in the study. Our results suggest that the probability of developing hemangiosarcoma or one of the other included pathological conditions within 6-18 months of testing was less than 2% in dogs with a negative result (low risk), but >90% in dogs with a positive result (high risk). We will follow the dogs in this study for the duration of their lifetime to assess the predictive value of the blood test over time. Dogs at high risk of hemangiosarcoma are eligible to receive eBAT, a drug capable of eliminating the cells that maintain and propagate the tumor, while making the environment inhospitable for tumor growth. This will provide means to explore the safety and efficacy of eBAT in the setting of prevention. This study serves as proof of concept for a new paradigm of early detection and strategic prevention to reduce the societal impact of canine and human cancers. Citation Format: Jaime F. Modiano, Taylor A. DePauw, Ali Khammanivong, Ashley J. Schulte, Amber L. Winter, Jong Kim, Kathleen Stuebner, Andrea Fahrenkrug, Daniel A. Vallera, Antonella Borgatti, Erin B. Dickerson, Michael S. Henson. Early detection for strategic prevention of a terminal canine cancer: A model to reduce the impact of cancer in our society [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4592.

Research paper thumbnail of The adrenergic receptor antagonists propranolol and carvedilol decrease bone sarcoma cell viability and sustained carvedilol reduces clonogenic survival and increases radiosensitivity in canine osteosarcoma cells

Veterinary and Comparative Oncology

Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metasta... more Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metastatic tumor growth and may influence proliferation, survival, metastasis, and angiogenesis. AR antagonists like propranolol and carvedilol inhibit proliferation, induce apoptosis, and synergize with chemotherapy agents in some cancers. Radiation resistance is mediated in many cells by upregulation of pro-survival pathways, which may be influenced by ARs. Studies evaluating AR antagonists combined with radiation are limited. The purpose of this study was to determine the effect of propranolol and carvedilol on viability and radiosensitivity in sarcoma cell lines. The hypothesis was that propranolol and carvedilol would increase radiosensitivity in four primary bone sarcoma cell lines. Single agent propranolol or carvedilol inhibited cell viability in all cell lines in a concentration-dependent manner. The mean inhibitory concentrations (IC50 ) for carvedilol were approximately 4-fold lower than propranolol and may be clinically relevant in vivo. Immunoblot analysis confirmed AR expression in both human and canine sarcoma cell lines; however, there was no correlation between baseline AR protein expression and radiosensitivity. Short duration treatment with carvedilol and propranolol did not significantly affect clonogenic survival. Prolonged exposure to propranolol and carvedilol significantly decreased the surviving fraction of canine osteosarcoma cells after 3Gy radiation. Based on our results and possible in vivo activity in dogs, further studies investigating the effects of carvedilol on sarcoma are warranted. This article is protected by copyright. All rights reserved.

Research paper thumbnail of A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumor growth of feline oral squamous cell carcinoma

Veterinary and Comparative Oncology

Monocarboxylate transporters (MCTs) support tumor growth by regulating the transport of metabolit... more Monocarboxylate transporters (MCTs) support tumor growth by regulating the transport of metabolites in the tumor microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistance malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 killed feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism, and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumor growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Intracellular calprotectin (S100A8/A9) controls epithelial differentiation and caspase-mediated cleavage of EGFR in head and neck squamous cell carcinoma

Oral Oncology

OBJECTIVES Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck sq... more OBJECTIVES Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck squamous cell carcinoma (HNSCC) and expression in the carcinoma cells and patient survival rates are directly related. We seek to characterize the suppressive role of calprotectin in HNSCC. AIMS (1) Investigate changes in S100A8/A9 expression as oral carcinogenesis progresses and (2) determine whether intracellular calprotectin can regulate epidermal growth factor receptor (EGFR), a negative prognostic factor, in HNSCC. MATERIALS AND METHODS Using immunohistochemistry (IHC), S100A8/A9 was analyzed in HNSCC specimens (N = 46), including well-differentiated (WD, N = 19), moderately-differentiated (MD, N = 14), poorly-differentiated (PD, N = 5) and non-keratinizing/basaloid (NK/BAS, N = 8), and premalignant epithelial dysplasias (PED, N = 16). Similarly, EGFR was analyzed in HNSCCs (N = 21). To determine whether calprotectin and EGFR expression are mechanistically linked, TR146 HNSCC cells that are S100A8/A9-expressing or silenced (shRNA) were compared for EGFR levels and caspase-3/7 activity using western blotting and immunofluorescence microscopy. RESULTS In normal oral mucosal epithelium, S100A8/A9 stained strongly in the cytoplasm and nucleus of suprabasal cells; basal cells were consistently S100A8/A9 negative. In PED and HNSCC, S100A8/A9 expression was lower than in adjacent normal epithelial tissues (NAT) and declined progressively in WD, MD, PD and NK/BAS HNSCCs. S100A8/A9 and EGFR levels appeared inversely related, which was simulated in vitro when S100A8/A9 was silenced in TR146 cells. Silencing S100A8/A9 significantly reduced caspase-3/7 activity, whereas EGFR levels increased. CONCLUSIONS In HNSCC, S100A8/A9 is directly associated with cellular differentiation and appears to promote caspase-3/7-mediated cleavage of EGFR, which could explain why patients with S100A8/A9-high tumors survive longer.

Research paper thumbnail of Abstract LB-013: Propranolol alters the intracellular accumulation of doxorubicin and sensitizes angiosarcoma cells to chemotherapy

Experimental and Molecular Therapeutics

Research paper thumbnail of Abstract 2886: CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma

Cancer Research

Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, wher... more Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, where treatments often result in high rates of failure and disease recurrence. A subpopulation of tumor-initiating cancer stem cells (CSC) is thought to be responsible for metastatic invasion and drug resistance in many types of cancer, including HNSCC. CD44 is a known CSC marker in HNSCC but its role in maintaining CSC populations is not well understood. We previously reported that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) inhibition of bone morphogenetic protein (BMP) signaling is essential for maintaining a CD44-high CSC-like population in HNSCC. In this study, we sought to determine how CD44, a receptor for hyaluronic acid (HA) that constitutes a major component of the tumor stroma, is involved in the regulation of SMURF1 and the maintenance of a HNSCC CSC phenotype. CSC-like cells were enriched from HNSCC cell lines as CD44-high cells. CD44 signaling was stimulated by exogenous HA treatment and inhibited by CD44 knockout (KO) using an inducible CRISPR/Cas9 system. We then assessed changes in SMURF1 protein level, BMP signaling, transwell migration, Matrigel colony formation, and invasion through three-dimensional organotypic culture (OTC) following CD44 modulation. CD44-high cells were found to have increased extracellular HA production. Treatment with exogenous HA reduced BMP signaling, as determined by a reduction in phospho-SMAD1/5/8 levels, and increased transwell migration of CD44-high cells. CD44-KO reduced SMURF1 protein expression and inhibited Matrigel colony formation of an invasive and recurrent HNSCC-derived CD44-high cells but only partially reduced colony formation of a less-invasive cell line derived from a primary HNSCC. Knockout of CD44 expression showed a slight reduction in transwell migration of invasive cells. CD44-high cells also recapitulated an invasive and CSC-like growth pattern in OTC assays. In contrast, CD44-KO inhibited OTC invasion and the epithelial-to-mesenchymal transition (EMT) phenotype, resulting in increased apical epithelial growth and differentiation. CD44 reconstitution appeared to restore the invasive and EMT phenotype. Based on our current findings, CD44 may be crucial for maintaining an anchorage-independent and invasive phenotype of HNSCC but plays a minor role in carcinoma cell migration. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2017-2886

Research paper thumbnail of Use of non-selective β-blockers is associated with decreased tumor proliferative indices in early stage breast cancer

Oncotarget, Jan 23, 2016

Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast... more Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of β1-AR and β3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of β-blocker usage on tumor proliferation. Our analysis revealed that non-selective β-blockers, but not selective β-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of β-blockers were measured in a pan...

Research paper thumbnail of Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis

for their support and for reviewing and making my final thesis defense possible. Furthermore, I r... more for their support and for reviewing and making my final thesis defense possible. Furthermore, I respectfully extend my appreciation to the current and past members of the Herzberg Laboratory for all their support and contribution. Firstly, I acknowledge Dr. Chengxing Wang (deceased) for his work on the mouse tumor model and for initiating the study of calprotectin in cancer growth regulation; and Anne Jernberg for her work on the in vivo tumor growth study. Secondly, I want to thank Dr. Brian Guenther and Brent Sorenson for their support and critical reviews of my proposal and manuscript throughout the entire thesis project; and especially Brent for his contribution to the gene silencing studies in TR146 HNSCC cells. I thank Min Gyu Kwak for his assistance in the protein expression, PP2A phosphatase signaling and matrix metalloproteinase studies; and Kevin Castleberry for his contribution to the calprotectin-PP2A protein-protein interaction study; Jing (Grace) Tian for her assistance in the real-time RT-PCR gene expression analysis; and Timothy Jernberg for his help with the calprotectin gene knockdown in TR146 cells. Lastly, I acknowledge Dr.

Research paper thumbnail of Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC

Oncotarget, Jan 13, 2016

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and ... more Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongl...

Research paper thumbnail of Transcriptome profiling in oral cavity and esophagus tissues from ( S )-N′-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis

Molecular Carcinogenesis, 2016

Recently, we have shown that (S)-N′-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacc... more Recently, we have shown that (S)-N′-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 weeks and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate p-value < 0.05 and fold-change ≥ 2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4 and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1 and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80 and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and therefore could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers.

Research paper thumbnail of Abstract 1950: SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells (CSCs) po... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, and tumor initiation thought to be responsible for drug resistance and treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Previously, we showed that the activity of bone morphogenetic protein (BMP), a key regulator of cell differentiation, proliferation, migration, and apoptosis, is inhibited in CSC-like populations enriched from HNSCC cell lines. In contrast, the BMP signaling inhibitor SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) and CD44, a marker of CSCs, were upregulated. Objectives: In this study, we characterized the role of SMURF1 for CSC maintenance and differentiating growth and identified a population of cells regulated by SMURF1. Methods: We generated a stable knockdown of SMURF1 expression in CSC-like populations using a lentivirus delivered shRNA interference construct. Long-term colony formation and Calcein AM dye efflux assays were performed to assess CSC maintenance and differentiating growth. Results: Knockdown of SMURF1 expression by shRNA reduced viability and colony formation of CSC-like cells grown in three-dimensional culture. Furthermore, reduced SMURF1 expression diminished the number of cells capable of dye efflux and decreased CD44 expression (CD44-high) in a select cell population. Conclusion: We interpret our data to suggest that SMURF1 inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs. Reduced dye efflux and expression of CD44-high cells suggests decreased maintenance of CSC-like populations along with an increased potential for differentiating growth. Targeting SMURF1 may provide a long-term benefit for HNSCC treatment to reduce resistant CSC populations and disease recurrence. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1950. doi:10.1158/1538-7445.AM2014-1950

Research paper thumbnail of SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma

Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, ... more Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, and tumor initiation thought to be responsible for drug resistance and treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Previously, we showed that the activity of bone morphogenetic protein (BMP), a key regulator of cell differentiation, proliferation, migration, and apoptosis, is inhibited in CSC-like populations enriched from HNSCC cell lines. In contrast, the BMP signaling inhibitor SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) and CD44, a marker of CSCs, were upregulated. Objectives: In this study, we characterized the role of SMURF1 for CSC maintenance and differentiating growth and identified a population of cells regulated by SMURF1. Methods: We generated a stable knockdown of SMURF1 expression in CSC-like populations using a lentivirus delivered shRNA interference construct. Long-term colony formation and Calcein AM dye eff...

Research paper thumbnail of Calprotectin Negatively Regulates Head and Neck Squamous Cell Carcinoma

Calprotectin is a heterodimer of S100A8 (MRP8) and S100A9 (MRP14) calcium-binding proteins normal... more Calprotectin is a heterodimer of S100A8 (MRP8) and S100A9 (MRP14) calcium-binding proteins normally expressed in the cytoplasm of squamous epithelial cells, but is down-regulated in head and neck squamous cell carcinoma (HNSCC). Generally functioning as an innate immune response molecule, calprotectin was recently found to control tumor growth at the G2/M cell cycle checkpoint. When in complex with S100A8, the C-terminal region of the S100A9 subunit binds to arachidonic acid (AA), a precursor of prostaglandin E2 (PGE2) biosynthesis implicated in malignant transformation. Objectives: We sought to determine whether calprotectin regulates malignant transformation-associated genes and if the C-terminal AA binding domain of S100A9 in calprotectin affects suppression of tumor growth and invasion in HNSCC. Methods: Calprotectin-negative and calprotectin-transfected oral epithelial carcinoma cell lines were compared for gene expression profiles using Affymetrix human genome array chip HU133...

Research paper thumbnail of Content Alerts

This article cites 38 articles, 19 of which can be accessed free

Research paper thumbnail of Molecular Cancer

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. SMURF1 silencing diminishes a CD44-high cancer stem cell-like population in

Research paper thumbnail of Abstract 671: Development of an exosomal biomarker signature to detect minimal residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning

Clinical Research (Excluding Clinical Trials)

Research paper thumbnail of Propranolol Sensitizes Vascular Sarcoma Cells to Doxorubicin by Altering Lysosomal Drug Sequestration and Drug Efflux

Frontiers in Oncology

Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few... more Angiosarcoma is a rare cancer of blood vessel–forming cells with a high patient mortality and few treatment options. Although chemotherapy often produces initial clinical responses, outcomes remain poor, largely due to the development of drug resistance. We previously identified a subset of doxorubicin-resistant cells in human angiosarcoma and canine hemangiosarcoma cell lines that exhibit high lysosomal accumulation of doxorubicin. Hydrophobic, weak base chemotherapeutics, like doxorubicin, are known to sequester within lysosomes, promoting resistance by limiting drug accessibility to cellular targets. Drug synergy between the beta adrenergic receptor (β-AR) antagonist, propranolol, and multiple chemotherapeutics has been documented in vitro, and clinical data have corroborated the increased therapeutic potential of propranolol with chemotherapy in angiosarcoma patients. Because propranolol is also a weak base and accumulates in lysosomes, we sought to determine whether propranolol...

Research paper thumbnail of Abstract B22: A method for early detection of hemangiosarcoma in dogs

Poster Presentations - Proffered Abstracts

Research paper thumbnail of Abstract B59: Validation of an exosomal osteosarcoma-associated gene signature in dogs with osteosarcoma

Poster Presentations - Proffered Abstracts

Research paper thumbnail of Abstract 1845: Propranolol reduces hemangiosarcoma cell viability by disrupting lipid homeostatic pathways

Experimental and Molecular Therapeutics

Research paper thumbnail of Abstract 4592: Early detection for strategic prevention of a terminal canine cancer: A model to reduce the impact of cancer in our society

Molecular and Cellular Biology / Genetics

Cancer mortality has surpassed all other causes of human death in parts of the developed world. D... more Cancer mortality has surpassed all other causes of human death in parts of the developed world. Domestic dogs and cats are the only other species where cancer has an equivalent impact in overall mortality. Rapid gains in longevity over the past two centuries are sufficient to explain the increased incidence of cancer in all three species. Not only does longevity increase the probability of exposures to environmental mutagens, but it also allows for introduction of mutations in each somatic cell replication. In this context, an increase in the incidence of cancer becomes almost inevitable because the evolutionary, cancer protective adaptations of humans, dogs, and cats are insufficient to provide protection over the 2 to 6-fold extension in lifespan achieved by these species. We believe this problem is addressable by developing tests to detect malignancies at their earliest stages and pairing these tests with strategic interventions to eliminate tumors before they form. To test this premise, we have developed a blood test to detect the presence of hemangiosarcoma in dogs, a rapidly fatal malignancy. The shorter lifespan of dogs, compared to humans, and the similar shorter latency of disease provide an ideal opportunity to assess the feasibility of our approach. The test uses flow cytometry for detection of rare, non-leukocyte nucleated cells in blood, combining 42 parameters for analysis in a machine learning environment. Samples from 126 dogs with a confirmed diagnosis of hemangiosarcoma (n=28); other cancers (n=29); benign vascular pathology of the spleen (n=27); or apparently healthy and under 4 years of age (n=41) were used for training and 10-fold cross validation. The expected sensitivity and specificity for the diagnosis of hemangiosarcoma in this training set were approximately 90% and 95%, respectively. We then used our trained models to assign a risk category to apparently healthy dogs, 6 years of age or older, from three breeds with a predisposition for hemangiosarcoma (up to 20% lifetime risk): samples from 105 golden retrievers, 52 boxers and 52 Portuguese Water Dogs (N=209) were included in the study. Our results suggest that the probability of developing hemangiosarcoma or one of the other included pathological conditions within 6-18 months of testing was less than 2% in dogs with a negative result (low risk), but >90% in dogs with a positive result (high risk). We will follow the dogs in this study for the duration of their lifetime to assess the predictive value of the blood test over time. Dogs at high risk of hemangiosarcoma are eligible to receive eBAT, a drug capable of eliminating the cells that maintain and propagate the tumor, while making the environment inhospitable for tumor growth. This will provide means to explore the safety and efficacy of eBAT in the setting of prevention. This study serves as proof of concept for a new paradigm of early detection and strategic prevention to reduce the societal impact of canine and human cancers. Citation Format: Jaime F. Modiano, Taylor A. DePauw, Ali Khammanivong, Ashley J. Schulte, Amber L. Winter, Jong Kim, Kathleen Stuebner, Andrea Fahrenkrug, Daniel A. Vallera, Antonella Borgatti, Erin B. Dickerson, Michael S. Henson. Early detection for strategic prevention of a terminal canine cancer: A model to reduce the impact of cancer in our society [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4592.

Research paper thumbnail of The adrenergic receptor antagonists propranolol and carvedilol decrease bone sarcoma cell viability and sustained carvedilol reduces clonogenic survival and increases radiosensitivity in canine osteosarcoma cells

Veterinary and Comparative Oncology

Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metasta... more Adrenergic receptor (AR) expression has been demonstrated at several sites of primary and metastatic tumor growth and may influence proliferation, survival, metastasis, and angiogenesis. AR antagonists like propranolol and carvedilol inhibit proliferation, induce apoptosis, and synergize with chemotherapy agents in some cancers. Radiation resistance is mediated in many cells by upregulation of pro-survival pathways, which may be influenced by ARs. Studies evaluating AR antagonists combined with radiation are limited. The purpose of this study was to determine the effect of propranolol and carvedilol on viability and radiosensitivity in sarcoma cell lines. The hypothesis was that propranolol and carvedilol would increase radiosensitivity in four primary bone sarcoma cell lines. Single agent propranolol or carvedilol inhibited cell viability in all cell lines in a concentration-dependent manner. The mean inhibitory concentrations (IC50 ) for carvedilol were approximately 4-fold lower than propranolol and may be clinically relevant in vivo. Immunoblot analysis confirmed AR expression in both human and canine sarcoma cell lines; however, there was no correlation between baseline AR protein expression and radiosensitivity. Short duration treatment with carvedilol and propranolol did not significantly affect clonogenic survival. Prolonged exposure to propranolol and carvedilol significantly decreased the surviving fraction of canine osteosarcoma cells after 3Gy radiation. Based on our results and possible in vivo activity in dogs, further studies investigating the effects of carvedilol on sarcoma are warranted. This article is protected by copyright. All rights reserved.

Research paper thumbnail of A novel MCT1 and MCT4 dual inhibitor reduces mitochondrial metabolism and inhibits tumor growth of feline oral squamous cell carcinoma

Veterinary and Comparative Oncology

Monocarboxylate transporters (MCTs) support tumor growth by regulating the transport of metabolit... more Monocarboxylate transporters (MCTs) support tumor growth by regulating the transport of metabolites in the tumor microenvironment. High MCT1 or MCT4 expression is correlated with poor outcomes in human patients with head and neck squamous cell carcinoma (HNSCC). Recently, drugs targeting these transporters have been developed and may prove to be an effective treatment strategy for HNSCC. Feline oral squamous cell carcinoma (OSCC) is an aggressive and treatment-resistance malignancy resembling advanced or recurrent HNSCC. The goals of this study were to investigate the effects of a previously characterized dual MCT1 and MCT4 inhibitor, MD-1, in OSCC as a novel treatment approach for feline oral cancer. We also sought to determine the potential of feline OSCC as a large animal model for the further development of MCT inhibitors to treat human HNSCC. In vitro, MD-1 killed feline OSCC and human HNSCC cell lines, altered glycolytic and mitochondrial metabolism, and synergized with platinum-based chemotherapies. While MD-1 treatment increased lactate concentrations in an HNSCC cell line, the inhibitor failed to alter lactate levels in feline OSCC cells, suggesting an MCT-independent activity. In vivo, MD-1 significantly inhibited tumor growth in a subcutaneous xenograft model and prolonged overall survival in an orthotopic model of feline OSCC. Our results show that MD-1 may be an effective therapy for the treatment of feline oral cancer. Our findings also support the further investigation of feline OSCC as a large animal model to inform the development of MCT inhibitors and future clinical studies in human HNSCC. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Intracellular calprotectin (S100A8/A9) controls epithelial differentiation and caspase-mediated cleavage of EGFR in head and neck squamous cell carcinoma

Oral Oncology

OBJECTIVES Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck sq... more OBJECTIVES Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck squamous cell carcinoma (HNSCC) and expression in the carcinoma cells and patient survival rates are directly related. We seek to characterize the suppressive role of calprotectin in HNSCC. AIMS (1) Investigate changes in S100A8/A9 expression as oral carcinogenesis progresses and (2) determine whether intracellular calprotectin can regulate epidermal growth factor receptor (EGFR), a negative prognostic factor, in HNSCC. MATERIALS AND METHODS Using immunohistochemistry (IHC), S100A8/A9 was analyzed in HNSCC specimens (N = 46), including well-differentiated (WD, N = 19), moderately-differentiated (MD, N = 14), poorly-differentiated (PD, N = 5) and non-keratinizing/basaloid (NK/BAS, N = 8), and premalignant epithelial dysplasias (PED, N = 16). Similarly, EGFR was analyzed in HNSCCs (N = 21). To determine whether calprotectin and EGFR expression are mechanistically linked, TR146 HNSCC cells that are S100A8/A9-expressing or silenced (shRNA) were compared for EGFR levels and caspase-3/7 activity using western blotting and immunofluorescence microscopy. RESULTS In normal oral mucosal epithelium, S100A8/A9 stained strongly in the cytoplasm and nucleus of suprabasal cells; basal cells were consistently S100A8/A9 negative. In PED and HNSCC, S100A8/A9 expression was lower than in adjacent normal epithelial tissues (NAT) and declined progressively in WD, MD, PD and NK/BAS HNSCCs. S100A8/A9 and EGFR levels appeared inversely related, which was simulated in vitro when S100A8/A9 was silenced in TR146 cells. Silencing S100A8/A9 significantly reduced caspase-3/7 activity, whereas EGFR levels increased. CONCLUSIONS In HNSCC, S100A8/A9 is directly associated with cellular differentiation and appears to promote caspase-3/7-mediated cleavage of EGFR, which could explain why patients with S100A8/A9-high tumors survive longer.

Research paper thumbnail of Abstract LB-013: Propranolol alters the intracellular accumulation of doxorubicin and sensitizes angiosarcoma cells to chemotherapy

Experimental and Molecular Therapeutics

Research paper thumbnail of Abstract 2886: CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma

Cancer Research

Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, wher... more Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, where treatments often result in high rates of failure and disease recurrence. A subpopulation of tumor-initiating cancer stem cells (CSC) is thought to be responsible for metastatic invasion and drug resistance in many types of cancer, including HNSCC. CD44 is a known CSC marker in HNSCC but its role in maintaining CSC populations is not well understood. We previously reported that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) inhibition of bone morphogenetic protein (BMP) signaling is essential for maintaining a CD44-high CSC-like population in HNSCC. In this study, we sought to determine how CD44, a receptor for hyaluronic acid (HA) that constitutes a major component of the tumor stroma, is involved in the regulation of SMURF1 and the maintenance of a HNSCC CSC phenotype. CSC-like cells were enriched from HNSCC cell lines as CD44-high cells. CD44 signaling was stimulated by exogenous HA treatment and inhibited by CD44 knockout (KO) using an inducible CRISPR/Cas9 system. We then assessed changes in SMURF1 protein level, BMP signaling, transwell migration, Matrigel colony formation, and invasion through three-dimensional organotypic culture (OTC) following CD44 modulation. CD44-high cells were found to have increased extracellular HA production. Treatment with exogenous HA reduced BMP signaling, as determined by a reduction in phospho-SMAD1/5/8 levels, and increased transwell migration of CD44-high cells. CD44-KO reduced SMURF1 protein expression and inhibited Matrigel colony formation of an invasive and recurrent HNSCC-derived CD44-high cells but only partially reduced colony formation of a less-invasive cell line derived from a primary HNSCC. Knockout of CD44 expression showed a slight reduction in transwell migration of invasive cells. CD44-high cells also recapitulated an invasive and CSC-like growth pattern in OTC assays. In contrast, CD44-KO inhibited OTC invasion and the epithelial-to-mesenchymal transition (EMT) phenotype, resulting in increased apical epithelial growth and differentiation. CD44 reconstitution appeared to restore the invasive and EMT phenotype. Based on our current findings, CD44 may be crucial for maintaining an anchorage-independent and invasive phenotype of HNSCC but plays a minor role in carcinoma cell migration. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2017-2886

Research paper thumbnail of Use of non-selective β-blockers is associated with decreased tumor proliferative indices in early stage breast cancer

Oncotarget, Jan 23, 2016

Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast... more Previous studies suggest beta-adrenergic receptor (β-AR) antagonists (β-blockers) decrease breast cancer progression, tumor metastasis, and patient mortality; however the mechanism for this is unknown. Immunohistochemical analysis of normal and malignant breast tissue revealed overexpression of β1-AR and β3-AR in breast cancer. A retrospective cross-sectional study of 404 breast cancer patients was performed to determine the effect of β-blocker usage on tumor proliferation. Our analysis revealed that non-selective β-blockers, but not selective β-blockers, reduced tumor proliferation by 66% (p < 0.0001) in early stage breast cancer compared to non-users. We tested the efficacy of propranolol on an early stage breast cancer patient, and quantified the tumor proliferative index before and after treatment, revealing a propranolol-mediated 23% reduction (p = 0.02) in Ki67 positive tumor cells over a three-week period. The anti-proliferative effects of β-blockers were measured in a pan...

Research paper thumbnail of Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis

for their support and for reviewing and making my final thesis defense possible. Furthermore, I r... more for their support and for reviewing and making my final thesis defense possible. Furthermore, I respectfully extend my appreciation to the current and past members of the Herzberg Laboratory for all their support and contribution. Firstly, I acknowledge Dr. Chengxing Wang (deceased) for his work on the mouse tumor model and for initiating the study of calprotectin in cancer growth regulation; and Anne Jernberg for her work on the in vivo tumor growth study. Secondly, I want to thank Dr. Brian Guenther and Brent Sorenson for their support and critical reviews of my proposal and manuscript throughout the entire thesis project; and especially Brent for his contribution to the gene silencing studies in TR146 HNSCC cells. I thank Min Gyu Kwak for his assistance in the protein expression, PP2A phosphatase signaling and matrix metalloproteinase studies; and Kevin Castleberry for his contribution to the calprotectin-PP2A protein-protein interaction study; Jing (Grace) Tian for her assistance in the real-time RT-PCR gene expression analysis; and Timothy Jernberg for his help with the calprotectin gene knockdown in TR146 cells. Lastly, I acknowledge Dr.

Research paper thumbnail of Involvement of calprotectin (S100A8/A9) in molecular pathways associated with HNSCC

Oncotarget, Jan 13, 2016

Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and ... more Calprotectin (S100A8/A9), a heterodimeric protein complex of calcium-binding proteins S100A8 and S100A9, plays key roles in cell cycle regulation and inflammation, with potential functions in squamous cell differentiation. While upregulated in many cancers, S100A8/A9 is downregulated in squamous cell carcinomas of the cervix, esophagus, and the head and neck (HNSCC). We previously reported that ectopic S100A8/A9 expression inhibits cell cycle progression in carcinoma cells. Here, we show that declining expression of S100A8/A9 in patients with HNSCC is associated with increased DNA methylation, less differentiated tumors, and reduced overall survival. Upon ectopic over-expression of S100A8/A9, the cancer phenotype of S100A8/A9-negative carcinoma cells was suppressed in vitro and tumor growth in vivo was significantly decreased. MMP1, INHBA, FST, LAMC2, CCL3, SULF1, and SLC16A1 were significantly upregulated in HNSCC but were downregulated by S100A8/A9 expression. Our findings strongl...

Research paper thumbnail of Transcriptome profiling in oral cavity and esophagus tissues from ( S )-N′-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis

Molecular Carcinogenesis, 2016

Recently, we have shown that (S)-N′-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacc... more Recently, we have shown that (S)-N′-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 weeks and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate p-value < 0.05 and fold-change ≥ 2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4 and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1 and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80 and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and therefore could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers.

Research paper thumbnail of Abstract 1950: SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma

Cancer Research, 2014

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells (CSCs) po... more Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, and tumor initiation thought to be responsible for drug resistance and treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Previously, we showed that the activity of bone morphogenetic protein (BMP), a key regulator of cell differentiation, proliferation, migration, and apoptosis, is inhibited in CSC-like populations enriched from HNSCC cell lines. In contrast, the BMP signaling inhibitor SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) and CD44, a marker of CSCs, were upregulated. Objectives: In this study, we characterized the role of SMURF1 for CSC maintenance and differentiating growth and identified a population of cells regulated by SMURF1. Methods: We generated a stable knockdown of SMURF1 expression in CSC-like populations using a lentivirus delivered shRNA interference construct. Long-term colony formation and Calcein AM dye efflux assays were performed to assess CSC maintenance and differentiating growth. Results: Knockdown of SMURF1 expression by shRNA reduced viability and colony formation of CSC-like cells grown in three-dimensional culture. Furthermore, reduced SMURF1 expression diminished the number of cells capable of dye efflux and decreased CD44 expression (CD44-high) in a select cell population. Conclusion: We interpret our data to suggest that SMURF1 inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs. Reduced dye efflux and expression of CD44-high cells suggests decreased maintenance of CSC-like populations along with an increased potential for differentiating growth. Targeting SMURF1 may provide a long-term benefit for HNSCC treatment to reduce resistant CSC populations and disease recurrence. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1950. doi:10.1158/1538-7445.AM2014-1950

Research paper thumbnail of SMURF1 inhibition reduces cancer stem cell-like population in head and neck squamous cell carcinoma

Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, ... more Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal, and tumor initiation thought to be responsible for drug resistance and treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Previously, we showed that the activity of bone morphogenetic protein (BMP), a key regulator of cell differentiation, proliferation, migration, and apoptosis, is inhibited in CSC-like populations enriched from HNSCC cell lines. In contrast, the BMP signaling inhibitor SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) and CD44, a marker of CSCs, were upregulated. Objectives: In this study, we characterized the role of SMURF1 for CSC maintenance and differentiating growth and identified a population of cells regulated by SMURF1. Methods: We generated a stable knockdown of SMURF1 expression in CSC-like populations using a lentivirus delivered shRNA interference construct. Long-term colony formation and Calcein AM dye eff...

Research paper thumbnail of Calprotectin Negatively Regulates Head and Neck Squamous Cell Carcinoma

Calprotectin is a heterodimer of S100A8 (MRP8) and S100A9 (MRP14) calcium-binding proteins normal... more Calprotectin is a heterodimer of S100A8 (MRP8) and S100A9 (MRP14) calcium-binding proteins normally expressed in the cytoplasm of squamous epithelial cells, but is down-regulated in head and neck squamous cell carcinoma (HNSCC). Generally functioning as an innate immune response molecule, calprotectin was recently found to control tumor growth at the G2/M cell cycle checkpoint. When in complex with S100A8, the C-terminal region of the S100A9 subunit binds to arachidonic acid (AA), a precursor of prostaglandin E2 (PGE2) biosynthesis implicated in malignant transformation. Objectives: We sought to determine whether calprotectin regulates malignant transformation-associated genes and if the C-terminal AA binding domain of S100A9 in calprotectin affects suppression of tumor growth and invasion in HNSCC. Methods: Calprotectin-negative and calprotectin-transfected oral epithelial carcinoma cell lines were compared for gene expression profiles using Affymetrix human genome array chip HU133...