Carmen Z Michaylira

Publications of Carmen Z Michaylira

• Periostin, a cell adhesion molecule, facilitates invasion in the tumor microenvironment and annotates a novel tumor-invasive signature in esophageal cancer.

  Authors: Carmen Z Michaylira, Gabrielle S Wong, Charles G Miller, Christie M Gutierrez, Hiroshi Nakagawa, Rachel Hammond, Andres J Klein-Szanto, Ju-Seog Lee, Sang Bae Kim, Meenhard Herlyn, J Alan Diehl, Phyllis Gimotty, Anil K Rustgi

  Cancer research. 07/2010; 70(13):5281-92.

  Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate that isHuman squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate that is related to a propensity for invasion and metastasis. Here, we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor-invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor-invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain-of-loss and loss-of-function experiments. Inhibition of epidermal growth factor receptor signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting the interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (three-dimensional) culture can offer an important tool to discover novel biological effectors in cancer.

• Fibroblast-secreted hepatocyte growth factor plays a functional role in esophageal squamous cell carcinoma invasion.

  Authors: Katharine D Grugan, Charles G Miller, Yao Yao, Carmen Z Michaylira, Shinya Ohashi, Andres J Klein-Szanto, J Alan Diehl, Meenhard Herlyn, May Han, Hiroshi Nakagawa, Anil K Rustgi

  Proceedings of the National Academy of Sciences of the United States of America. 06/2010; 107(24):11026-31.

  Squamous cell cancers comprise the most common type of human epithelial cancers. One subtype, esophageal squamous cell carcinoma (ESCC), is an aggressive cancer with poor prognosis due to lateSquamous cell cancers comprise the most common type of human epithelial cancers. One subtype, esophageal squamous cell carcinoma (ESCC), is an aggressive cancer with poor prognosis due to late diagnosis and metastasis. Factors derived from the extracellular matrix (ECM) create an environment conducive to tumor growth and invasion. Specialized cancer-associated fibroblasts (CAFs) in the ECM influence tumorigenesis. We have shown previously that the nature and activation state of fibroblasts are critical in modulating the invasive ability of ESCC in an in vivo-like organotypic 3D cell culture, a form of human tissue engineering. Dramatic differences in invasion of transformed esophageal epithelial cells depended on the type of fibroblast in the matrix. We hypothesize that CAFs create an environment primed for growth and invasion through the secretion of factors. We find that fibroblast secretion of hepatocyte growth factor (HGF) fosters the ability of transformed esophageal epithelial cells to invade into the ECM, although other unidentified factors may cooperate with HGF. Genetic modifications of both HGF in fibroblasts and its receptor Met in epithelial cells, along with pharmacologic inhibition of HGF and Met, underscore the importance of this pathway in ESCC invasion and progression. Furthermore, Met activation is increased upon combinatorial overexpression of epidermal growth factor receptor (EGFR) and p53(R175H), two common genetic mutations in ESCC. These results highlight the potential benefit of the therapeutic targeting of HGF/Met signaling in ESCC and potentially other squamous cancers where this pathway is deregulated.

• Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

  Authors: Shinya Ohashi, Mitsuteru Natsuizaka, Gabrielle S Wong, Carmen Z Michaylira, Katharine D Grugan, Douglas B Stairs, Jiri Kalabis, Maria E Vega, Ross A Kalman, Momo Nakagawa, Andres J Klein-Szanto, Meenhard Herlyn, J Alan Diehl, Anil K Rustgi, Hiroshi Nakagawa

  Cancer research. 05/2010; 70(10):4174-84.

  Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellularTransforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

• Hypoxia activates the cyclooxygenase-2-prostaglandin E synthase axis.

  Authors: James J Lee, Mitsuteru Natsuizaka, Shinya Ohashi, Gabrielle S Wong, Munenori Takaoka, Carmen Z Michaylira, Daniela Budo, John W Tobias, Michiyuki Kanai, Yasuhiro Shirakawa, Yoshio Naomoto, Andres J P Klein-Szanto, Volker H Haase, Hiroshi Nakagawa

  Carcinogenesis. 03/2010; 31(3):427-34.

  Hypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expressionHypoxia-inducible factors (HIFs), in particular HIF-1alpha, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1alpha target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1alpha by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1alpha. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E(2) (PGE(2)) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE(2) production in a HIF-1alpha-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1alpha and IGFBP3. Activation of the COX-2-PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1beta and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1alpha target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin.

• Cyclin D1 overexpression increases susceptibility to 4-nitroquinoline-1-oxide-induced dysplasia and neoplasia in murine squamous oral epithelium.

  Authors: Jonathan F Wilkey, Glenn Buchberger, Kirsten Saucier, Salony M Patel, Ellen Eisenberg, Hiroshi Nakagawa, Carmen Z Michaylira, Anil K Rustgi, Sanjay M Mallya

  Molecular carcinogenesis. 04/2009;

  The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue,The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus, and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect of cyclin D1 overexpression on susceptibility of mice to carcinogen-induced tumorigenesis, using 4-nitroquinoline-1-oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and nontransgenic littermates were administered 4NQO (20 or 50 parts per million (ppm) in the drinking water) for 8 wk and monitored for an additional 16 wk. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with nontransgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro-esophageal epithelia. Examination of the dysplastic and neoplastic lesions revealed abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen-induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero-digestive cancer. (c) 2009 Wiley-Liss, Inc.

• A subpopulation of mouse esophageal basal cells has properties of stem cells with the capacity for self-renewal and lineage specification.

  Authors: Jiri Kalabis, Kenji Oyama, Takaomi Okawa, Hiroshi Nakagawa, Carmen Z Michaylira, Douglas B Stairs, Jose-Luiz Figueiredo, Umar Mahmood, J Alan Diehl, Meenhard Herlyn, Anil K Rustgi

  The Journal of clinical investigation. 01/2009; 118(12):3860-9.

  The esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, theyThe esophageal epithelium is a prototypical stratified squamous epithelium that exhibits an exquisite equilibrium between proliferation and differentiation. After basal cells proliferate, they migrate outward toward the luminal surface, undergo differentiation, and eventually slough due to apoptosis. The identification and characterization of stem cells responsible for the maintenance of the esophageal epithelium remains elusive. Here, we employed Hoechst dye extrusion and BrdU label-retaining assays to identify in mice a potential esophageal stem cell population that localizes to the basal cell compartment. The self-renewing capacity of this population was characterized using a clonogenic assay and a 3D organotypic culture model. The putative esophageal stem cells were also capable of epithelial reconstitution in vivo in direct esophageal epithelial injury models. In both the 3D organotypic culture and direct mucosal injury models, the putative stem cells gave rise to undifferentiated and differentiated cells. These studies therefore provide a basis for understanding the regenerative capacity and biology of the esophageal epithelium when it is faced with injurious insults.

• The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation.

  Authors: Takaomi Okawa, Carmen Z Michaylira, Jiri Kalabis, Douglas B Stairs, Hiroshi Nakagawa, Claudia D Andl, Cameron N Johnstone, Andres J Klein-Szanto, Wafik S El-Deiry, Edna Cukierman, Meenhard Herlyn, Anil K Rustgi

  Genes & development. 12/2007; 21(21):2788-803.

  Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform toEsophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.

• IGFBP-3 regulates esophageal tumor growth through IGF-dependent and independent mechanisms.

  Authors: Munenori Takaoka, Seok Hyun Kim, Takaomi Okawa, Carmen Z Michaylira, Douglas B Stairs, Cameron N Johnstone, Claudia D Andl, Ben Rhoades, James J Lee, Andres J P Klein-Szanto, Wafik S El-Deiry, Hiroshi Nakagawa

  Cancer biology & therapy. 05/2007; 6(4):534-40.

  Insulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophagealInsulin-like growth factor binding protein (IGFBP)-3 exerts either proapoptotic or growth stimulatory effects depending upon the cellular context. IGFBP-3 is overexpressed frequently in esophageal cancer. Yet, the role of IGFBP-3 in esophageal tumor biology remains elusive. To delineate the functional consequences of IGFBP-3 overexpression, we stably transduced Ha-Ras(V12)-transformed human esophageal cells with either wild-type or mutant IGFBP-3, the latter incapable of binding Insulin-like growth factor (IGFs) as a result of substitution of amino-terminal Ile56, Leu80, and Leu81 residues with Glycine residues. Wild-type, but not mutant, IGFBP-3 prevented IGF-1 from activating the IGF-1 receptor and AKT, and suppressed anchorage-independent cell growth. When xenografted in nude mice, in vivo bioluminescence imaging demonstrated that wild-type, but not mutant IGFBP-3, abrogated tumor formation by the Ras-transformed cells with concurrent induction of apoptosis, implying a prosurvival effect of IGF in cancer cell adaptation to the microenvironment. Moreover, there was more aggressive tumor growth by mutant IGFBP-3 overexpressing cells than control cell tumors, without detectable caspase-3 cleavage in tumor tissues, indicating an IGF-independent growth stimulatory effect of mutant IGFBP-3. In aggregate, these data suggest that IGFBP-3 contributes to esophageal tumor development and progression through IGF-dependent and independent mechanisms.

• Hypoxic microenvironment as a cradle for melanoma development and progression.

  Authors: Carmen Z Michaylira, Hiroshi Nakagawa

  Cancer biology & therapy. 06/2006; 5(5):476-9.

  Hypoxia-inducible factor (HIF)-1alpha, a global regulator of oxygen homeostasis, plays a crucial role in tumor cell adaptation to the hypoxic microenvironment through transcriptional regulation ofHypoxia-inducible factor (HIF)-1alpha, a global regulator of oxygen homeostasis, plays a crucial role in tumor cell adaptation to the hypoxic microenvironment through transcriptional regulation of its target genes. These genes in turn are involved in a plethora of biochemical as well as cell biological processes, including glucose metabolism, apoptosis and angiogenesis. In melanoma, HIF-1alpha has been implicated in tumor progression with effects upon metastasis and angiogenesis. However, its role in malignant transformation by oncogenes has not been described. Bedogni et al. (Cancer Cell 2005, 8:443-54) report that the hypoxic microenvironment in the skin contributes to melanocyte transformation and tumor growth induced by oncogenes Ras and Akt, which are frequently activated in melanoma. HIF-1alpha activity was found to be required in Akt-induced melanocyte transformation and tumor growth and it was suppressed greatly by mTOR inhibition with rapamycin. Since mTOR regulates HIF-1alpha expression and its transcriptional activity, rapamycin was proposed as a promising hypoxia-related therapeutic approach in melanoma treatment. This study sheds light upon the role of HIF-1alpha in the early stage of melanoma development and highlights the importance of the Akt-mTOR pathway in the regulation of HIF-1alpha.