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1.  Identification of secreted proteins that reflect autophagy dynamics within tumor cells 
Autophagy  2014;11(1):60-74.
Macroautophagy, a catabolic process of cellular self-digestion, is an important tumor cell survival mechanism and a potential target in antineoplastic therapies. Recent discoveries have implicated autophagy in the cellular secretory process, but potential roles of autophagy-mediated secretion in modifying the tumor microenvironment are poorly understood. Furthermore, efforts to inhibit autophagy in clinical trials have been hampered by suboptimal methods to quantitatively measure tumor autophagy levels. Here, we leveraged the autophagy-based involvement in cellular secretion to identify shed proteins associated with autophagy levels in melanoma. The secretome of low-autophagy WM793 melanoma cells was compared to its highly autophagic metastatic derivative, 1205Lu in physiological 3-dimensional cell culture using quantitative proteomics. These comparisons identified candidate autophagy biomarkers IL1B (interleukin 1, β), CXCL8 (chemokine (C-X-C motif) ligand 8), LIF (leukemia inhibitory factor), FAM3C (family with sequence similarity 3, member C), and DKK3 (dickkopf WNT signaling pathway inhibitor 3) with known roles in inflammation and tumorigenesis, and these proteins were subsequently shown to be elevated in supernatants of an independent panel of high-autophagy melanoma cell lines. Secretion levels of these proteins increased when low-autophagy melanoma cells were treated with the autophagy-inducing tat-BECN1 (Beclin 1) peptide and decreased when ATG7 (autophagy-related 7) was silenced in high-autophagy cells, thereby supporting a mechanistic link between these secreted proteins and autophagy. In addition, serum from metastatic melanoma patients with high tumor autophagy levels exhibited higher levels of these proteins than serum from patients with low-autophagy tumors. These results suggest that autophagy-related secretion affects the tumor microenvironment and measurement of autophagy-associated secreted proteins in plasma and possibly in tumors can serve as surrogates for intracellular autophagy dynamics in tumor cells.
PMCID: PMC4502670  PMID: 25484078
autophagy; biomarker; cancer; melanoma; secretome; ATG5, autophagy-related 5; ATG7, autophagy-related 7; BECN1, Beclin 1, autophagy-related; AV, autophagic vacuole; CXCL8, chemokine (C-X-C motif) ligand 8; DKK3, dickkopf WNT signaling pathway inhibitor 3; EGF, epidermal growth factor; IF, interstitial fluid; IL1B, interleukin 1, β; LC3/MAP1LC3, microtubule-associated protein 1 light chain 3; LIF, leukemia inhibitory factor; M, media; PtdIns3K, phosphatidylinositol 3-kinase; SAM, significance analysis of microarrays
2.  Cellular senescence checkpoint function determines differential Notch1-dependent oncogenic and tumor suppressor activities 
Oncogene  2014;34(18):2347-2359.
Notch activity regulates tumor biology in a context-dependent and complex manner. Notch may act as an oncogene or a tumor suppressor gene even within the same tumor type. Recently, Notch signaling has been implicated in cellular senescence. Yet, it remains unclear as to how cellular senescence checkpoint functions may interact with Notch-mediated oncogenic and tumor suppressor activities. Herein, we used genetically engineered human esophageal keratinocytes and esophageal squamous cell carcinoma cells to delineate the functional consequences of Notch activation and inhibition along with pharmacological intervention and RNA interference (RNAi) experiments. When expressed in a tetracycline-inducible manner, the ectopically expressed activated form of Notch1 (ICN1) displayed oncogene-like characteristics inducing cellular senescence corroborated by the induction of G0/G1 cell-cycle arrest, Rb dephosphorylation, flat and enlarged cell morphology and senescence-associated β-galactosidase activity. Notch-induced senescence involves canonical CSL/RBPJ-dependent transcriptional activity and the p16INK4A-Rb pathway. Loss of p16INK4A or the presence of human papilloma virus (HPV) E6/E7 oncogene products not only prevented ICN1 from inducing senescence, but permitted ICN1 to facilitate anchorage-independent colony formation and xenograft tumor growth with increased cell proliferation and reduced squamous-cell differentiation. Moreover, Notch1 appears to mediate replicative senescence as well as TGF-β-induced cellular senescence in non-transformed cells and that HPV E6/E7 targets Notch1 for inactivation to prevent senescence, revealing a tumor suppressor attribute of endogenous Notch1. In aggregate, cellular senescence checkpoint functions may influence dichotomous Notch activities in the neoplastic context.
PMCID: PMC4268095  PMID: 24931169
Notch; Rb; p16; HPV; E7; senescence; squamous cell carcinoma
3.  MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines 
Oncogene  2013;33(14):1850-1861.
Elevated activity of the MAPK signaling cascade is found in the majority of human melanomas and is known to regulate proliferation, survival, and invasion. Current targeted therapies focus on decreasing the activity of this pathway; however, we do not fully understand how these therapies impact tumor biology, especially given that melanoma is a heterogeneous disease. Using a three-dimensional (3D), collagen-embedded spheroid melanoma model, we observed that MEK and BRAF inhibitors can increase the invasive potential of approximately 20% of human melanoma cell lines. The invasive cell lines displayed increased receptor tyrosine kinase (RTK) activity and activation of the Src/FAK/STAT3 signaling axis, also associated with increased cell-to-cell adhesion and cadherin engagement following MEK inhibition. Targeting various RTKs, Src, FAK, and STAT3 with small molecule inhibitors in combination with a MEK inhibitor prevented the invasive phenotype, but only STAT3 inhibition caused cell death in the 3D context. We further show that STAT3 signaling is induced in BRAF-inhibitor resistant cells. Our findings suggest that MEK and BRAF inhibitors can induce STAT3 signaling, causing potential adverse effects such as increased invasion. We also provide the rationale for the combined targeting of the MAPK pathway along with inhibitors of RTKs, SRC, or STAT3 to counteract STAT3-mediated resistance phenotypes.
PMCID: PMC3769503  PMID: 23624919
melanoma; MEK; STAT3; targeted therapy; invasion; resistance
4.  Prognosis of Patients with Melanoma and Microsatellitosis Undergoing Sentinel Lymph Node Biopsy 
Annals of surgical oncology  2013;21(3):1016-1023.
Melanoma microsatellitosis is classified as stage IIIB/C disease and is associated with a poor prognosis. Prognostic factors within this group, however, have not been well characterized.
We performed a retrospective analysis of 1621 patients undergoing sentinel lymph node (SLN) biopsy at our institution (1996–2011) to compare patients with (n=98) and patients without (n=1523) microsatellites. Univariate and multivariate logistic and Cox regression analyses were used to identify factors associated with SLN positivity and melanoma-specific survival (MSS) in patients with microsatellites.
Patients with microsatellites were older and had lesions with higher Clark level and greater thickness that more frequently had mitoses, ulceration, and lymphovascular invasion (LVI) (all p<0.0001). In microsatellite patients, the SLN positivity rate was 43%. Lesional ulceration (OR=2.9, 95% CI: 1.5–8.6), absent tumor infiltrating lymphocytes (OR=2.8, 95% CI: 1.1–7.1), and LVI (OR=3.3, 95% CI: 1.7–10.0) were significantly associated with SLN positivity by multivariate analysis. With a median follow up of 4.5 years in survivors, ulceration (HR=3.4, 95% CI: 1.5–7.8) and >1 metastatic LN (HR=2.7, 95% CI: 1.1–6.6) were significantly associated with decreased MSS by multivariate analysis. In patients without these prognostic factors, the 5-year MSS was 90% (n=49), compared to 50% (n=23) among patients with ulceration only, 51% (n=12) in those with >1 metastatic LN only, or 25% in those with both (n=14, p<0.01).
Microsatellitosis was frequently associated with multiple adverse pathologic features. In the absence of ulceration and >1 metastatic LN, however, the outcome for patients with microsatellites compared favorably to stage IIIB patients overall.
PMCID: PMC4001246  PMID: 24258854
5.  EGFR Inhibition Promotes an Aggressive Invasion Pattern Mediated by Mesenchymal-like Tumor Cells within Squamous Cell Carcinomas 
Molecular cancer therapeutics  2013;12(10):2176-2186.
Squamous cell carcinomas (SCCs) with an infiltrative invasion pattern carry a higher risk of treatment failure. Such infiltrative invasion may be mediated by a mesenchymal-like subpopulation of malignant cells that we have previously shown to arise from epithelial to mesenchymal transition (EMT) and resist epidermal growth factor receptor (EGFR) targeting. Here we demonstrate that SCCs with infiltrative, high risk invasion patterns contain abundant mesenchymal-like cells, which are rare in tumors with low risk patterns. This cellular heterogeneity was modeled accurately in three dimensional culture using collagen-embedded SCC spheroids, which revealed distinct invasive fronts created by collective migration of E-cadherin-positive cells versus infiltrative migration of individual mesenchymal-like cells. Because EGFR expression by mesenchymal-like cells was diminished in the spheroid model and in human SCCs, we hypothesized that SCCs shift toward infiltrative invasion mediated by this subpopulation during anti-EGFR therapy. Anti-EGFR treatment of spheroids using erlotinib or cetuximab enhanced infiltrative invasion by targeting collective migration by E-cadherin-positive cells while sparing mesenchymal-like cells; by contrast, spheroid invasion in absence of mesenchymal-like cells was abrogated by erlotinib. Similarly, cetuximab treatment of xenografts containing mesenchymal-like cells created an infiltrative invasive front comprised of this subpopulation, whereas no such shift was observed upon treating xenografts lacking these cells. These results implicate mesenchymal-like SCC cells as key mediators of the infiltrative invasion seen in tumors with locally aggressive behavior. They further demonstrate that EGFR inhibition can promote an infiltrative invasion front comprised of mesenchymal-like cells preferentially in tumors where they are abundant prior to therapy.
PMCID: PMC3796003  PMID: 23939378
pattern of invasion; EGFR inhibition; squamous cell carcinoma; EMT; tumor heterogeneity
6.  Age as a predictor of sentinel node metastasis among patients with localized melanoma 
Annals of surgical oncology  2014;21(4):1075-1081.
We have previously reported that older patients with clinical Stage I and II primary cutaneous. melanoma had lower survival rates compared to younger patients We postulated that the incidence of nodal metastasis would therefore be higher among older melanoma patients.
Materials and Methods
The expanded AJCC Melanoma Staging Database contains a cohort of 7756 melanoma patients who presented without clinical evidence of regional lymph node or distant metastasis and who underwent a sentinel node biopsy procedure as a component of their staging workup
Although older patients had primary melanoma features associated with more aggressive biology, we observed paradoxically a significant decrease in the incidence of sentinel node metastasis as patient age increased. Overall, the highest incidence of sentinel node metastasis was 25.8% in patients under 20 years of age, compared to 15.5% in patients 80 years and older (p< 0.001). In contrast, five year mortality rates for Clinical Stage II patients ranged from a low of 20% for those 20–40 years of age up to 38% for those over 70 years of age. Patient age was an independent predictor of sentinel node metastasis in a multifactorial analysis (p<0.001)
Patients with clinical Stage I and II melanoma under 20 years of age had a higher incidence of sentinel lymph node metastasis but, paradoxically, a more favorable survival outcome compared to all other age groups. In contrast, patients >70 years had the most aggressive primary melanoma features and a higher mortality rate compared to all other age groups, but a lower incidence of sentinel lymph node metastasis.
We analyzed the importance of patient age as a component of melanoma staging and in the design and interpretation of clinical trials for patients with localized disease or with regional node metastases.
PMCID: PMC4121329  PMID: 24531700
7.  Age as a Prognostic Factor in Patients with Localized Melanoma and Regional Metastases 
Annals of surgical oncology  2013;20(12):3961-3968.
We postulated that the worse prognosis of melanoma with advancing age reflected more aggressive tumor biology and that in younger patients the prognosis would be more favorable.
Materials and Methods
The expanded AJCC melanoma staging database contained 11,088 patients with complete data for analysis, including mitotic rate.
With increasing age by decade, primary melanomas were thicker, exhibited higher mitotic rates, and were more likely to be ulcerated. In a multivariate analysis of patients with localized melanoma, thickness and ulceration were highly significant predictors of outcome at all decades of life (except for patients less than 20 years). Mitotic rate was significantly predictive in all age groups except patients < 20 years and >80 years. For patients with Stage III melanoma, there were four independent variables associated with patient survival: number of nodal metastases, patient age, ulceration, and mitotic rate.
Patients under 20 years of age had primary tumors with slightly more aggressive features, a higher incidence of sentinel lymph node metastasis, but, paradoxically, more favorable survival than all other age groups. In contrast, patients >70 years old had primary melanomas with the most aggressive prognostic features, were more likely to be head and neck primaries, and were associated with a higher mortality rate than the other age groups. Surprisingly, however, these patients had a lower rate of sentinel lymph node metastasis per T stage. Among patients between the two age extremes, clinicopathologic features and survival tended to be more homogeneous.
Melanomas in patients at the extremes of age have a distinct natural history.
PMCID: PMC4121330  PMID: 23838920
8.  Overcoming intrinsic multi-drug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1Bhigh cells 
Cancer cell  2013;23(6):10.1016/j.ccr.2013.05.003.
Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.
PMCID: PMC3810180  PMID: 23764003
9.  Effect of Guidelines on Primary Care Physician Use of PSA Screening: Results from the Community Tracking Study Physician Survey 
Little is known about the effect of guidelines that recommend shared decision making on physician practice patterns. The objective of this study was to determine the association between physicians’ perceived effect of guidelines on clinical practice and self-reported prostate-specific antigen (PSA) screening patterns.
This was a cross-sectional study using a nationally representative sample of 3914 primary care physicians participating in the 1998–1999 Community Tracking Study Physician Survey. Responses to a case vignette that asked physicians what proportion of asymptomatic 60-year-old white men they would screen with a PSA were divided into 3 distinct groups: consistent PSA screeners (screen all), variable screeners (screen 1%–99%), and consistent nonscreeners (screen none). Logistic regression was used to determine the association between PSA screening patterns and physician-reported effect of guidelines (no effect v. any magnitude effect).
Only 27% of physicians were variable PSA screeners; the rest were consistent screeners (60%) and consistent nonscreeners (13%). Only 8% of physicians perceived guidelines to have no effect on their practice. After adjustment for demographic and practice characteristics, variable screeners were more likely to report any magnitude effect of guidelines on their practice when compared with physicians in the other 2 groups (adjusted odds ratio 1.73; 95% confidence interval = 1.25–2.38; P = 0.001).
Physicians who perceive an effect of guidelines on their practice are almost twice as likely to exhibit screening PSA practice variability, whereas physicians who do not perceive an effect of guidelines on their practice are more likely to be consistent PSA screeners or consistent PSA nonscreeners.
PMCID: PMC3991564  PMID: 18556635
prostate-specific antigen; mass screening; guidelines; physicians’ practice patterns
10.  Optical Imaging of Periostin Enables Early Endoscopic Detection and Characterization of Esophageal Cancer in Mice 
Gastroenterology  2012;144(2):294-297.
Imaging strategies that detect early-stage esophageal squamous cell carcinoma (ESCC) could improve clinical outcomes, combined with endoscopic approaches. Periostin is an integrin-binding protein that is important in the tumor microenvironment. We created a fluorescent-labeled antibody that recognizes periostin and binds specifically to ESCC xenograft tumors in mice. In L2-cre;p120ctnLoxP/LoxP mice, which develop squamous cell cancers that resemble human ESCC, we visualized the probe in preneoplastic and neoplastic esophageal lesions using near-infrared fluorescent imaging with upper gastrointestinal endoscopy. Periostin might be a biomarker of the esophageal tumor microenvironment that can be used to detect preneoplastic lesions.
PMCID: PMC3624041  PMID: 23085486
mouse model; neoplasm; extracellular matrix; POSTN
11.  IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment 
Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.
PMCID: PMC3902230  PMID: 24482736
CD44; esophageal; squamous cell carcinoma; hypoxia; IGFBP3 and reactive oxygen species
12.  Acquired cancer stem cell phenotypes through Oct4-mediated dedifferentiation 
Oncogene  2012;31(47):4898-4911.
There is enormous interest to target cancer stem cells (CSCs) for clinical treatment because these cells are highly tumorigenic and resistant to chemotherapy. Oct4 is expressed by CSC-like cells in different types of cancer. However, function of Oct4 in tumor cells is unclear. In this study, we showed that expression of Oct4 gene or transmembrane delivery of Oct4 protein promoted dedifferentiation of melanoma cells to CSC-like cells. The dedifferentiated melanoma cells showed significantly decreased expression of melanocytic markers and acquired the ability to form tumor spheroids. They showed markedly increased resistance to chemotherapeutic agents and hypoxic injury. In the subcutaneous xenograft and tail vein injection assays, these cells had significantly increased tumorigenic capacity. The dedifferentiated melanoma cells acquired features associated with CSCs such as multipotent differentiation capacity and expression of melanoma CSC markers such as ABCB5 and CD271. Mechanistically, Oct4 induced dedifferentiation was associated with increased expression of endogenous Oct4, Nanog and Klf4, and global gene expression changes that enriched for transcription factors. RNAi mediated knockdown of Oct4 in dedifferentiated cells led to diminished CSC phenotypes. Oct4 expression in melanoma was regulated by hypoxia and its expression was detected in a subpopulation of melanoma cells in clinical samples. Our data indicate that Oct4 is a positive regulator of tumor dedifferentiation. The results suggest that CSC phenotype is dynamic and may be acquired through dedifferentiation. Oct4 mediated tumor cell dedifferentiation may play an important role during tumor progression.
PMCID: PMC3343184  PMID: 22286766
Oct4; Cancer stem cell; Melanoma; Dedifferentiation; hypoxia
13.  Direct reprogramming of melanocytes to neural crest stem-like cells by one defined factor 
Stem cells (Dayton, Ohio)  2011;29(11):1752-1762.
Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest stem-like cells. These induced neural crest stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express neural crest-related genes and differentiate into multiple neural crest derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in ovo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive neural crest cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications.
PMCID: PMC3615703  PMID: 21948558
Notch; melanocytes; neural crest stem cells; reprogramming; dedifferentiation
14.  A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer 
Journal of Proteome Research  2011;11(2):678-691.
Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients.
PMCID: PMC3272104  PMID: 22032327
Biomarker discovery; ovarian cancer; cancer biomarkers; xenograft mouse model; proteomics; multidimensional protein profiling
15.  Lymphatic Invasion is Independently Prognostic of Metastasis in Primary Cutaneous Melanoma 
Clinical Cancer Research  2011;18(1):229-237.
Lymphatic invasion (LI) in primary cutaneous melanomas was recently found to be common. In this study, we evaluated LI as an independent prognostic factor.
Experimental Design
This study included 251 patients with vertical growth phase (VGP) primary cutaneous melanomas who had paraffin-fixed lesional tissue and were in a prospective cohort seen between 1972 and 1991, had no clinical evidence of regional nodal disease at diagnosis and had at least ten years of follow-up. Dual immunohistochemistry (IHC) staining was used to detect lymphatic endothelium (podoplanin) and melanoma cells (S-100). Multivariate logistic regression for ten-year metastasis was used to define independent prognostic factors and a prognostic tree was developed to characterize and discriminate risk groups. Kaplan-Meier disese-free survival curves for those with and without LI within current AJCC stages were compared using the log-rank statistic.
LI was observed in 43% (108 of 251) of the study melanomas. The multivariate model for ten-year metastasis identified 4 independent prognostic factors: tumor thickness, mitotic rate (MR), LI, and anatomic site. The prognostic tree identified a group of patients with thin (≤1 mm thick) melanomas and poor prognosis: stage IB melanomas with LI. Survival curves for time to first metastasis demonstrated significantly poorer prognosis for patients with LI compared to those without it for both stages IB and IIA.
LI is common across the range of tumor thicknesses in primary VGP melanomas. It is an independent prognostic factor and significantly increases the risk of metastasis in patients in clinical stages IB and IIA.
PMCID: PMC3262994  PMID: 22096024
16.  A NOTCH3-mediated squamous cell differentiation program limits expansion of EMT competent cells that express the ZEB transcription factors 
Cancer research  2011;71(21):6836-6847.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
PMCID: PMC3206139  PMID: 21890822
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
17.  Genetic and Pharmacologic Inhibition of Complement Impairs Endothelial Cell Function and Ablates Ovarian Cancer Neovascularization1 
Neoplasia (New York, N.Y.)  2012;14(11):994-1004.
Complement activation plays a critical role in controlling inflammatory responses. To assess the role of complement during ovarian cancer progression, we crossed two strains of mice with genetic complement deficiencies with transgenic mice that develop epithelial ovarian cancer (TgMISIIR-TAg). TgMISIIR-TAg mice fully or partially deficient for complement factor 3 (C3) (Tg+C3KO and Tg+C3HET, respectively) or fully deficient for complement factor C5a receptor (C5aR) (Tg+C5aRKO) develop either no ovarian tumors or tumors that were small and poorly vascularized compared to wild-type littermates (Tg+C3WT, Tg+C5aRWT). The percentage of tumor infiltrating immune cells in Tg+C3HET tumors compared to Tg+C3WT controls was either similar (macrophages, B cells, myeloid-derived suppressor cells), elevated (effector T cells), or decreased (regulatory T cells). Regardless of these ratios, cytokine production by immune cells taken from Tg+C3HET tumors was reduced on stimulation compared to Tg+C3WT controls. Interestingly, CD31+ endothelial cell (EC) function in angiogenesis was significantly impaired in both C3KO and C5aRKO mice. Further, using the C5aR antagonist PMX53, tube formation of ECs was shown to be C5a-dependent, possibly through interactions with the VEGF165 but not VEGF121 isoform. Finally, the mouse VEGF164 transcript was underexpressed in C3KO livers compare to C3WT livers. Thus, we conclude that complement inhibition blocks tumor outgrowth by altering EC function and VEGF165 expression.
PMCID: PMC3514739  PMID: 23226093
18.  Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice 
Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected.
Methods and results
Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice.
Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.
PMCID: PMC3426288  PMID: 18157673
GD2; Peptide mimics; Antibodies; DTH; Tumor growth inhibition
19.  Tissue-based immune monitoring I 
Cancer Biology & Therapy  2011;12(4):357-366.
We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of seven consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8 and FoxP3. A portion of the cores was used to isolate RNA for (1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGFβ, (2) multiplexed PCR-based T-cell receptor (TCR) CDR3 V-β region spectratyping and (3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3-epsilon, CD8A and FoxP3 gene expression, respectively, by qPCR (r = 0.61, 0.86 and 0.89; all p < 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR V-β chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.
PMCID: PMC3173733  PMID: 21785264
ovarian neoplasms; lymphocytes; tumor-infiltrating; adoptive immunotherapy; gene expression profiling; immunohistochemistry
20.  Tissue-based immune monitoring II 
Cancer Biology & Therapy  2011;12(4):367-377.
The presence of tumor-infiltrating lymphocytes (TILs) in epithelial ovarian cancer indicates a host antitumor response and is associated with improved survival. We wished to determine the extent to which TIL density differs from site to site within a given patient. We initially studied multiple paired metastases from serous ovarian carcinoma obtained at the time of primary debulking. The expression of genes in specific immune-related pathways was profiled on a pilot set of five patients. We then used immunohistochemistry and quantitative PCR to estimate the density of CD3+, CD8+ and Foxp3+ TILs in these same tumors. To extend the findings to a larger cohort, we semiquantitatively measured intraepithelial and stromal TILs in a tissue microarray (TMA) containing both primary tumors and metastases from 50 patients. In the pilot group, genes related to antimicrobial signaling and TGFβ signaling showed between-site heterogeneity, whereas cytokines and antigen presentation transcripts were more homogeneous in any given patient. IHC and qPCR for T-cell markers were concordant. In the TMA cohort, two-way ANOVA showed that TIL heterogeneity between sites was present in some but not all patients. The stroma of extra-ovarian metastases showed significantly greater TIL infiltration than ovarian sites. A simulation showed that at clinically meaningful levels of precision, up to 3% of patients will be misclassified for intraepithelial TILs by a single biopsy. In conclusion, between-site heterogeneity exists in some patients with metastatic serous ovarian cancer. The predictive value of biopsies should be considered in clinical trial design.
PMCID: PMC3173734  PMID: 21785280
ovarian neoplasms; lymphocytes; tumor-infiltrating; adoptive immunotherapy; metastasis; microarray analysis
21.  Frequent genetic abnormalities of the PI3K/AKT pathway in primary ovarian cancer predict patient outcome 
Genes, chromosomes & cancer  2011;50(8):606-618.
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and /or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
PMCID: PMC3110626  PMID: 21563232
22.  Novel surface targets and serum biomarkers from the ovarian cancer vasculature 
Cancer Biology & Therapy  2011;12(3):169-180.
The molecular phenotype of tumor vasculature is different from normal vasculature, offering new opportunities for diagnosis and therapy of cancer, but the identification of tumor-restricted targets remains a challenge. We investigated 13 tumor vascular markers (TVMs) from 50 candidates identified through expression profiling of ovarian cancer vascular cells and selected to be either transmembrane or secreted, and to be either absent or expressed at low levels in normal tissues while overexpressed in tumors, based on analysis of 1,110 normal and tumor tissues from publicly available Affymetrix microarray data. Tumor-specific expression of each TVM was confirmed at the protein level in tumor tissue and/or in serum. Among the 13 TVMs, 11 were expressed on tumor vascular endothelium; the remaining 2 TVMs were expressed by tumor leukocytes. Our results demonstrate that certain transmembrane TVMs such as ADAM12 and CDCP1 are selectively expressed in tumor vasculature and represent promising targets for vascular imaging or anti-vascular therapy of epithelial ovarian cancer, while secreted or shed molecules such as TNFRSF21/DR6 can function as serum biomarkers. We have identified novel tumor-specific vasculature markers which appear promising for cancer serum diagnostics, molecular imaging and/or therapeutic targeting applications and warrant further clinical development.
PMCID: PMC3230481  PMID: 21617380
ovarian; cancer; vascular; biomarkers; diagnostics; serum; expression; profiling
23.  Notch receptor inhibition reveals the importance of cyclin D1 and Wnt signaling in invasive esophageal squamous cell carcinoma 
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
PMCID: PMC3410579  PMID: 22860235
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
24.  Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas 
Cell Cycle  2011;10(12):2008-2016.
Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44+CD24− stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.
PMCID: PMC3154419  PMID: 21558812
EMT; squamous cell carcinoma; head and neck cancer; esophageal cancer; chemotherapy resistance; salinomycin; tumor heterogeneity
25.  Prognostic Significance of Mitotic Rate in Localized Primary Cutaneous Melanoma: An Analysis of Patients in the Multi-Institutional American Joint Committee on Cancer Melanoma Staging Database 
Journal of Clinical Oncology  2011;29(16):2199-2205.
The aim of this study was to assess the independent prognostic value of primary tumor mitotic rate compared with other clinical and pathologic features of stages I and II melanoma.
From the American Joint Committee on Cancer (AJCC) melanoma staging database, information was extracted for 13,296 patients with stages I and II disease who had mitotic rate data available.
Survival times declined as mitotic rate increased. Ten-year survival ranged from 93% for patients whose tumors had 0 mitosis/mm2 to 48% for those with ≥ 20/mm2 (P < .001). Mean number of mitoses/mm2 increased as the primary melanomas became thicker (1.0 for melanomas ≤ 1 mm, 3.5 for 1.01 to 2.0 mm, 7.3 for 3.01 to 4.0 mm, and 9.6 for > 8 mm). Ulceration was also associated with a higher mitotic rate; 59% of ulcerated melanomas had ≥ 5 mitoses/mm2 compared with 16% of nonulcerated melanomas (P < .001). In a multivariate analysis of 10,233 patients, the independent predictive factors for survival in order of statistical significance were as follows: tumor thickness (χ2 = 104.9; P < .001), mitotic rate (χ2 = 67.0; P < .001), patient age (χ2 = 48.2; P < .001), ulceration (χ2 = 46.4; P < .001), anatomic site (χ2 = 34.6; P < .001), and patient sex (χ2 = 33.9; P < .001). Clark level of invasion was not an independent predictor of survival (χ2 = 3.2; P = .37).
A high mitotic rate in a primary melanoma is associated with a lower survival probability. Among the independent predictors of melanoma-specific survival, mitotic rate was the strongest prognostic factor after tumor thickness.
PMCID: PMC3107741  PMID: 21519009

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