Research Program
The Khavari Lab uses epithelial tissue as a model system to study stem cell biology, cancer and new molecular therapeutics. Epithelia cover external and internal body surfaces and undergo constant self-renewal while responding to diverse environmental stimuli. Epithelial homeostasis precisely balances stem cell-sustained proliferation and differentiation-associated cell death, a balance which is lost in many human diseases, including cancer. Epithelial neoplasms, including those affecting the skin, represent approximately 90% of all human malignancies.
Our experimental focus is on the mammalian setting for enhanced medical relevance. We use classical mouse genetics, human genetics and novel human tissue platforms. The latter encompass human skin regenerated on immune deficient mice as well as organotypic constructs with epithelial and stromal cells embedded within architecturally faithful mesenchymal tissue in vitro. These new models, which we group under the new term Multi-Functional Human Tissue Genetics, allow up to 10 alleles or more to be altered simultaneously, permitting genetic experiments with an unprecedented degree of rapidity and complexity exceeding that possible in classic genetic functionator organisms, such as yeast, worms and flies.
These new genetic models have facilitated the first conversions of normal human tissue into invasive cancer via alterations in defined genetic networks. For functional studies, we use an array of genetic interventions, including targeted gene disruption, RNA interference (RNAi) and conditionally active alleles, as well as pharmacologic and biochemical technologies. Bioinformatics-intensive systems biology methods are used alongside classical morphogenetic assessments to define critical regulators of homeostasis and neoplasia as a basis for targeted molecular therapeutics.
Stem cell biology and differentiation
In epithelial tissues, such as cutaneous epidermis, proliferative basal cells adherent to the underlying basement membrane undergo cell cycle arrest then outward migration and terminal differentiation. This process is mediated by two broad and mutually exclusive programs of gene expression: 1) an undifferentiated program supporting proliferation by stem cell progenitors within the basal layer and 2) a differentiation program instructing growth arrest and differentiation-associated programmed cell death in suprabasal layers. The control of this transition from epithelial stem cell to differentiated corneocyte, which is abnormal in epidermal cancers, is not well understood.
Our laboratory is currently pursuing studies of the signaling and gene regulatory networks that control this process. Among the most evolutionarily conserved signaling pathways in eukaryotes, the Ras/MAPK cascade involves Ras GTPase activation at cellular membranes followed by signal transmission down a MAPK kinase cascade to ultimately reprogram nuclear gene expression. Using murine and human genetic approaches, we recently demonstrated that maintenance of the undifferentiated stem cell state is dependent on intact Ras/MAPK signaling. Without Ras/MAPK function, epithelium loses proliferative self-renewal and undergoes irreversible terminal differentiation and cell death.
Studies to characterize the mechanisms responsible for Ras/MAPK pathway control of epithelial homeostasis, including identifications of genes regulating the pathway as well as end-stage effectors, are a focus of current efforts. Multiple signaling and gene regulatory networks interact with the Ras/MAPK cascade to control epithelial stem cell proliferation and differentiation, including NF-kappaB, the JNK cascade and core cell cycle regulators, such as Cdk4. Also included among these is the p53 transcription factor family member, p63, which is required for normal development of stratified epithelial tissues. We recently established that p63 acts dominantly within developmentally mature human epithelial tissue to control proliferation and differentiation in a cell autonomous manner. The mechanisms whereby p63 and other dominant regulators control cell cycle progression and induction of the differentiation program are an active area of investigation in the lab.
The Role of Epigenetic Mechanisms
In addition to studies of the classical signaling and gene regulatory networks noted above, we have recently identified a central role for additional biologic mechanisms, namely gene regulation by epigenetic mechanisms and by noncoding RNAs. Epigenetic control of gene expression lasts through multiple cell divisions without alterations in primary DNA sequence and can occur via mechanisms that include histone modification and DNA methylation. Noncoding RNA sequences can regulate gene expression via interactions with epigenetic and other control mechanisms. The function of epigenetic regulation and noncoding RNA as major regulators of epithelial stem cell renewal and differentiation represent major emerging areas of study in the lab.
Cancer
Epithelial cancers represent the vast majority of human malignancies and affect a wide range of body sites, including lung, colon, breast, prostate, ovary and skin. Skin malignancies, including epidermal squamous cell carcinoma (SCC), alone account for nearly as many cancers as all other tissues combined. SCC and other epithelial cancers develop through a process of tumor-stroma co-evolution in which epithelial cells undergo a stepwise progression towards invasive cancer in conjunction with remodeling of the underlying dermal stroma.
Progress in understanding epithelial carcinogenesis has been hindered in the past by a lack of models that faithfully recapitulate the three-dimensional architecture of tumor-stroma co-evolution. To address this and to also study the oncogenic potential of unregulated function of dominant regulators of epithelial homeostasis, such as the Ras/MAPK pathway, NF-kappaB and p63 noted above, we developed multiplex serial gene transfer (MSGT) in which alterations in 10 or more alleles can be made simultaneously. Combining MSGT with the capacity to regenerate human skin tissue with defined genetic alterations on immune deficient mice has permitted the molecular reconstruction of events sufficient to trigger human cancer and facilitated development of human tissue models of the three most common human skin malignancies, basal cell carcinoma, SCC and malignant melanoma. Grouped under the new term Multi-Functional Human Tissue Genetics, MFHTG is synergizing with new organotype models and sirna organonculeotide duplexes to form powerful new models.
These models are being used to systematically elucidate proteins required for cutaneous carcinogenesis and to test their potential role as therapeutic targets. Among these targets are extracellular proteins that mediate interactions between epithelial tumor cells and the surrounding stroma, including extracellular matrix collagen and laminin proteins and their integrin receptors. Targeting the function of specific molecular epitopes on these extracellular proteins, we are identifying promising therapeutic approaches designed to interrupt epithelial tumor progression.
Complementing these human tissue studies, we have pursued a genome-wide screening approach using laser capture microdissection and array-based transcript profiling to characterize genes expressed in different epidermal cancers and at specific steps in the transition from epithelial stem cell to growth arrested terminally differentiating cell. From these latter efforts, we have identified a host of previously uncharacterized genes altered in carcinogenesis that also appear part of precisely controlled genetic programs that accompany the growth arrest and differentiation process in epithelium. In combination, all of these studies are providing a broad perspective on the genetic controls of epithelial cell proliferation and are linking this information to human epithelial cancers.
Molecular Therapeutics
Epithelial tissues in general and skin in particular offer an attractive site for development of new approaches in molecular therapeutics. Our laboratory is currently pursuing development of new molecular therapeutics for the treatment of skin and systemic disease using novel human tissue-based models noted above. A family of human genetic skin diseases is characterized by defective epithelial gene expression. Among the most severe of these are subtypes of epidermolysis bullosa (EB) and lamellar ichthyosis (LI). We have developed approaches for high efficiency gene transfer to EB and LI patient skin tissue that are corrective at biochemical, histologic, clinical and functional levels. In addition to EB subtypes [LAMB3, BPAG2, COL7A1 genes] and LI [TGM1 gene], similar corrective efforts have also been undertaken with a number of other genetic skin disorders. These studies have been extended to refine new vectors capable of sustainable therapeutic gene delivery to epidermis as a basis for initiating clinical trials in humans.
In addition to these efforts at molecular correction of genetic skin disorders, new approaches for genetic vaccination and systemic gene delivery via the skin have also been established. Moreover, an array of nonviral and viral vectors has been used to achieve regulated delivery of therapeutic polypeptides to the bloodstream via cutaneous gene transfer. As a complement to gene-based therapeutics, we are studying new ways to introduce proteins and small molecule pharmacotherapeutics through the epidermal permeability barrier using conserved transporter domains. Finally, the potential of antibodies to alter cellular receptors and matrix interactions important in cancer are being analyzed using the capacity to develop genetically defined human cancers, as noted above. All of these efforts are aimed to advance progress in the application of molecular therapeutics to epithelial tissues.
