Aymee Perez

Muc4/sialomucin complex, the intramembrane Er6B2 ligand, in cancer and epithelia: To protect and to survive

The membrane mucin Muc4, also called sialomucin complex (SMC), is a heterodimeric complex of two subunits, ASGP-1 and ASGP-2, derived from a single gene. It is produced by multiple epithelia in both membrane and soluble forms and serves as a protective agent for the epithelia. The membrane form of Muc4 acts as a steric barrier to the apical cell surface of epithelial or tumor cells. An important example is the uterus of the rat, in which Muc4 expression is downregulated for blastocyst implantation. The soluble form facilitates the protection and lubrication of epithelia by mucous gels composed of gel-forming mucins, as in the airway, where Muc4 is proposed to participate in mucociliary transport as a constituent of the periciliary fluid. The soluble form is also found in body fluids, such as milk, tears, and saliva. The transmembrane subunit ASGP-2 acts as an intramembrane ligand and activator for the receptor tyrosine kinase ErbB2. Formation of this ligand-receptor complex is proposed to repress apopotosis in epithelial and cancer cells in which the ligand-receptor complex is formed, providing a second type of cell protective mechanism. Muc4 expression is regulated in epithelial tissues in a cell- and tissue-specific manner during epithelial differentiation. In stratified epithelia, it is predominantly in the most superficial, differentiated layers, often coincident with ErbB2. Dysregulation of Muc4 expression may contribute to cell and tissue dysfunction, such as the proposed contribution of Muc4 to mammary tumor progression. These observations clearly show that Muc4 has multiple roles in epithelia, which may provide insights into aberrant behaviors of these tissues and their derivative carcinomas.

Muc4/Sialomucin Complex in the Mammary Gland and Breast Cancer

MUC4 is a one of the membrane mucins of the mucin gene (MUC) family, characterized by mucin tandem repeat domains and a transmembrane domain which associates it with the cell plasma membrane. Although MUC4 is encoded by a single gene, it is produced by epithelial cells as a heterodimer through a proteolytic cleavage mechanism. This heterodimer is found in both membrane and soluble forms associated with epithelia. Functionally, MUC4 is proposed to provide a protective mechanism for vulnerable epithelia, such as those of the airway, eye, female reproductive tract and mammary gland. The protective mechanism(s) may be highjacked by some carcinomas, such as those of the breast, to increase tumor progression. Two mechanisms are proposed to contribute to the MUC4 functions. First, MUC4 acts as an anti-adhesive or anti-recognition barrier at epithelial or tumor cell surfaces. Second, MUC4 can bind the receptor tyrosine kinase ErbB2 and alter its cellular signaling. Expression of MUC4 in mammary gland is repressed by posttranscriptional mechanisms involving basement membrane and TGF-β, which are relieved during pregnancy to permit secretion of MUC4 into milk. These mechanisms are also abrogated in some breast cancers, providing a scenario for promotion of tumor progression. These observations imply important functions for MUC4 in both normal mammary function and in breast cancer.

CD44 interacts with EGFR and promotes head and neck squamous cell carcinoma initiation and progression

OBJECTIVES CD44 is a promising target for therapy in head and neck squamous cell carcinoma (HNSCC) and has two defined roles in tumorigenesis: it is a cancer stem cell (CSC) marker and it promotes migration and proliferation through interaction with many signaling molecules. The purpose of this study was to investigate the role of CD44 in HNSCC carcinogenesis. MATERIALS AND METHODS The effects of CD44 in cell proliferation, migration, apoptosis and cisplatin resistance were studied by its overexpression in HNSCC cells. We also evaluated the effect of CD44 on tumor progression by siRNA methodology, immunohistochemistry (IHC) and western blot analysis. CD44 and EGFR colocalization were examined in CAL 27 cells by laser scanning confocal microscopy. The interaction between CD44 and EGFR was analyzed by immunoprecipation. RESULTS Overexpression of CD44 enhances cell proliferation and migration and correlates with increased cisplatin resistance and apoptosis inhibition in SCC25 cells. Downregulation of CD44 in CAL27 cells inhibited constitutive EGFR phosphorylation and significantly reduced tumor growth in nude mice. CD44 and EGFR colocalized in CAL 27 cells. CD44 coimmunoprecipated with EGFR in CAL 27 cells, indicating that these proteins interact with each other. CONCLUSION CD44 therapy in HNSCC may target the CSC population and alter EGFR signaling.

Presence of MUC4 in human milk and at the luminal surfaces of blood vessels

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP‐1 (MUC4α) and a transmembrane subunit ASGP‐2 (MUC4β), which has been implicated in the protection of epithelial cell surfaces. Surprisingly, development and characterization of a new monoclonal antibody (mAb), called 1G8, against ASGP‐2 demonstrated by immunohistochemistry the presence of MUC4 at the luminal surfaces of blood vessels of both normal tissues and tumors. Muc4 was detected with 1G8 and other Muc4 antibodies in blood vessels from humans, rats and mice. This expression of MUC4 in endothelial cells was confirmed by immunoblotting with 1G8 in human umbilical vein endothelial cells (HUVECs), human iliac artery endothelial cells (HIAECs), and human microvascular endothelial cells (HMVECs). MUC4 could be observed on HUVECs grown on either plastic or Matrigel. Finally, MUC4 expression in the three types of endothelial cell lines was confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR). These results provide, to our knowledge, the first demonstration of a member of the MUC gene family and membrane mucin in blood vessels. As a luminal surface component, the MUC4 is situated to contribute to the non‐adhesive luminal surface and to act as an intrinsic protection and survival factor.

Salivary markers and risk factor data: A multivariate modeling approach for head and neck squamous cell carcinoma detection

BACKGROUND Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease largely due to late stage diagnosis. Prior work indicates that soluble CD44 (solCD44) and total protein may be useful diagnostic markers for HNSCC. In this study we combine the markers solCD44, IL-8, HA, and total protein with demographic and risk factor data to derive a multivariate logistic model that improves HNSCC detection as compared to our previous data using biomarkers alone. METHODS We performed the solCD44, IL-8, HA, and total protein assays on oral rinses from 40 HNSCC patients and 39 controls using ELISA assays. Controls had benign diseases of the upper aerodigestive tract and a history of tobacco or alcohol use. All subjects completed a questionnaire including demographic and risk factor data. RESULTS Depending on cancer subsite, differences between cases and controls were found for all markers. A multivariate logistic model including solCD44, total protein and variables related to smoking, oral health and education offered a significant improvement over the univariate models with an AUC of 0.853. Sensitivity ranged from 75-82.5% and specificity from 69.2-82.1% depending on predictive probability cut points. CONCLUSION A multivariate model, including simple and inexpensive molecular tests in combination with risk factors, results in a promising tool for distinguishing HNSCC patients from controls. IMPACT In this case-control study, the resulting observations led to an unprecedented multivariate model that distinguished HNSCC cases from controls with better accuracy than the current gold standard which includes oral examination followed by tissue biopsy. Since the components are simple, noninvasive, and inexpensive to obtain, this model combining biomarkers, risk factor and demographic data serves as a promising prototype for future cancer detection tests.

Abstract 2521: Targeting CD44 in head and neck squamous cell carcinoma (HNSCC) with a new humanized antibody RO5429083

Introduction: HNSCC is a debilitating and deadly disease for which current treatments are extremely toxic and ineffective. Increasing data suggest that persistent cancer stem cells (CSC), resistant to standard chemo and radiation therapy, give rise to disease recurrence. Thus, poor outcomes may reflect ineffective CSC killing. CD44 is a promising therapeutic target that has two defined roles in tumorigenesis: it is a putative cancer initiating cell or CSC marker and it promotes migration and proliferation through interaction with EGFR, Rho and Ras signaling. Therefore therapies that target CD44 may destroy the CSC population, which is resistant to therapy while precisely eradicating the proliferating population of cancer cells. Methods: RO5429083 is a novel functional monoclonal antibody, developed by Roche, that targets a glycosylated, conformation dependent epitope of CD44. To evaluate CD44 as a therapeutic target in vivo, we established CAL 27 (HNSCC cell line) xenografts in nude mice and treated them with RO5429083 (Roche) in comparison to cisplatin (DPP), a conventional drug used in HNSCC treatment. After treatment, the mice were sacrificed, and the tumors were embedded in paraffin for IHC analysis. We also analyzed the effect of RO5429083 in vitro on peripheral blood lymphocytes (PBMCs). Results: RO5429083 treatment displayed a remarkable tumor growth inhibition in CAL 27 xenografts compared to DPP or control (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2521. doi:1538-7445.AM2012-2521

Risk Stratification System for Oral Cancer Screening

Oral cavity and oropharyngeal cancer (oral cancer) is a deadly disease that is increasing in incidence. Worldwide 5-year survival is only 50% due to delayed intervention with more than half of the diagnoses at stage III and IV, whereas earlier detection (stage I and II) yields survival rates up to 80% to 90%. Salivary soluble CD44 (CD44), a tumor-initiating marker, and total protein levels may facilitate oral cancer risk assessment and early intervention. This study used a hospital-based design with 150 cases and 150 frequency-matched controls to determine whether CD44 and total protein levels in oral rinses were associated with oral cancer independent of age, gender, race, ethnicity, tobacco and alcohol use, and socioeconomic status (SES). High-risk subjects receiving oral cancer prevention interventions as part of a community-based program (n = 150) were followed over 1 year to determine marker specificity and variation. CD44 ≥5.33 ng/mL was highly associated with case status [adjusted OR 14.489; 95% confidence interval (CI), 5.973–35.145; P < .0001, vs. reference group CD44 <2.22 ng/mL and protein <1.23 mg/mL]. Total protein aided prediction above CD44 alone. Sensitivity and specificity in the frequency-matched study was 80% and 48.7%, respectively. However, controls were not representative of the target screening population due, in part, to a high rate of prior cancer. In contrast, specificity in the high-risk community was 74% and reached 95% after annual retesting. Simple and inexpensive salivary CD44 and total protein measurements may help identify individuals at heightened risk for oral cancer from the millions who partake in risky behaviors. Cancer Prev Res; 9(6); 445–55. ©2016 AACR.

A Case of AML Characterized by a Novel t(4;15)(q31;q22) Translocation That Confers a Growth-Stimulatory Response to Retinoid-Based Therapy

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.

A new approach to the treatment of acute myeloid leukaemia targeting the receptor for growth hormone-releasing hormone

Growth hormone‐releasing hormone (GHRH) is secreted by the hypothalamus and acts on the pituitary gland to stimulate the release of growth hormone (GH). GHRH can also be produced by human cancers, in which it functions as an autocrine/paracrine growth factor. We have previously shown that synthetic antagonistic analogues of GHRH are able to successfully suppress the growth of 60 different human cancer cell lines representing over 20 cancers. Nevertheless, the expression of GHRH and its receptors in leukaemias has never been examined. Our study demonstrates the presence of GHRH receptor (GHRH‐R) on 3 of 4 human acute myeloid leukaemia (AML) cell lines—K‐562, THP‐1, and KG‐1a—and significant inhibition of proliferation of these three cell lines in vitro following incubation with the GHRH antagonist MIA‐602. We further show that this inhibition of proliferation is associated with the upregulation of pro‐apoptotic genes and inhibition of Akt signalling in leukaemic cells. Treatment with MIA‐602 of mice bearing xenografts of these human AML cell lines drastically reduced tumour growth. The expression of GHRH‐R was further confirmed in 9 of 9 samples from patients with AML. These findings offer a new therapeutic approach to this malignancy and suggest a possible role of GHRH‐R signalling in the pathology of AML.

Abstract 3548: CD44, protein, demographics and risk factor data: A combined approach to detect head and neck squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease. The main risk factors are tobacco and alcohol use and human papillomavirus (HPV) infection. Early detection tests are needed because the majority of patients present in late stage when cure rates reach only 40%. Our group has developed a simple, inexpensive, noninvasive diagnostic test based on soluble CD44 (solCD44) and total protein levels. We used a case-control design to evaluate soluble markers for HNSCC in 150 oropharyngeal (OP) and lip/oral cavity (Lip/OC) patients and 150 controls frequency matched for age, gender, race, ethnicity, tobacco use and socioeconomic status. We compared patient groups with respect to important covariates using the chi-square, Fishers exact test, or t-test. Markers’ mean levels were compared either by t-test or ANOVA, followed by pairwise multiple comparisons. Logistic regression analysis was used to evaluate predictivity of the salivary markers univariately and multivariately with adjustment for demographics and risk factors. We report odds ratio (OR) estimates with corresponding 95% confidence interval (95% CI) and area under the curve (AUC) of the operating characteristic curve (ROC) for fitted models. The case-control groups did not differ in regards to age, gender, race, ethnicity, history of ever vs. never smoking, current alcohol use, number of teeth removed, or county vs. private hospital system. The mean log2CD44 and protein levels were elevated in cases (log2 CD44= 1.94 ng/ml, protein=0.93 mg/ml) compared to controls (log2 CD44= 1.28 ng/ml, protein= 0.76 mg/ml), (p<.0001; p=.003, respectively). Log2CD44 levels were significantly elevated in older vs. younger cases (p<0.05). There were no significant mean log2 CD44 level differences between Lip/OC and OP cases, TNM or HPV status in cancer patients. In the cases for which HPV status was available (as measured by the surrogate marker p16), log 2 CD44 levels varied by smoking status (lower in never smokers), by drinking status in HPV + cases (lower in non-drinkers) and N-stage (higher levels in N0, Nx vs. N1-N3 in HPV-positive and the opposite effect in HPV-negative tumors). For protein, there were no differences in either the case or the control group based on demographic or risk variables. When we stratified by HPV status, race/ethnicity and drinking status did have an effect (higher levels in blacks vs. WNH and higher in current vs. former and never/mild drinkers in HPV positive tumors only). A specific model was developed for each of the 3 race/ethnicity groups with the highest AUC for blacks (AUC= .835) and WNH (AUC= .831) followed by HW (AUC= .777). Models included log2CD44, protein, smoking, gender and age; strongly suggesting that these factors play an important role for HNSCC early detection studies. Citation Format: Lutecia H. Mateus Pereira, Isildinha Reis, Robert Duncan, Judy Wen, Erika Reategui, Stephanie Bayers, Laurian Walters, Aymee Perez, Jennifer Hu, W Jarrard Goodwin, Elizabeth J. Franzmann. CD44, protein, demographics and risk factor data: A combined approach to detect head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2013-3548