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Biomedical subjects

Paul A Khavari

Publications and source records attributed to Paul A Khavari.

At least 19 recordsLinked to original sources
PubMed2025

In Vivo CRISPR Interference Screen Reveals Long Noncoding RNA Portfolio Crucial for Cutaneous Squamous Cell Carcinoma Tumor Growth.

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of all skin cancer mortality globally, making it the second-highest subtype of skin cancer. The high prevalence of cSCC in humans highlights the need to uncover alternative actors and mechanisms influencing skin cancer development. Significant advances have been made to better understand some key factors in cSCC growth. However, little is known about the role of noncoding RNAs, particularly of a specific subclass termed long noncoding RNA (lncRNA). By performing pseudobulk analysis of single-cell sequencing data from normal and cSCC human skin tissues, we determined a global portfolio of lncRNAs specifically expressed in keratinocyte subpopulations. Integration of CRISPR interference screens in vitro and the xenograft model identified several lncRNAs impacting the growth of cSCC cancer lines both in vitro and in vivo. Among these, we further validated LINC00704 and LINC01116 as proliferation-regulating lncRNAs in cSCC lines and potential biomarkers of cSCC growth. Taken together, our study provides a comprehensive signature of lncRNAs with roles in regulating cSCC growth.

RNA, Long Noncoding↗
PubMed2025

Glucose modulates IRF6 transcription factor dimerization to enable epidermal differentiation.

Non-energetic roles for glucose are largely unclear, as is the interplay between transcription factors (TFs) and ubiquitous biomolecules. Metabolomic analyses uncovered elevation of intracellular glucose during differentiation of diverse cell types. Human and mouse tissue engineered with glucose sensors detected a glucose gradient that peaked in the outermost differentiated layers of the epidermis. Free glucose accumulation was essential for epidermal differentiation and required the SGLT1 glucose transporter. Glucose affinity chromatography uncovered glucose binding to diverse regulatory proteins, including the IRF6 TF. Direct glucose binding enabled IRF6 dimerization, DNA binding, genomic localization, and induction of IRF6 target genes, including essential pro-differentiation TFs GRHL1, GRHL3, HOPX, and PRDM1. These data identify a role for glucose as a gradient morphogen that modulates protein multimerization in cellular differentiation.

Cell Differentiation↗
PubMed2006

p63 regulates proliferation and differentiation of developmentally mature keratinocytes.

p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to down-regulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibited hypoproliferation, differentiation defects were not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescued the cell proliferation defect of p63 knockdown alone but failed to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Furthermore, DeltaNp63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes.

Cell Cycle↗
PubMed2006

Use of conditionally active ras fusion proteins to study epidermal growth, differentiation, and neoplasia.

Ras proteins are membrane-bound GTPases that play a central role in transmitting signals from the cell surface to the nucleus and affect a wide array of biological processes. The overall cellular response to Ras activation varies with cell type, experimental conditions, signal strength, and signal duration. Most current studies, however, rely on expression of constitutively active protein to study Ras function and thus ignore temporal variables, as well as signal strength. These experiments may provide contradictory results, as seen in the case of epidermal keratinocytes. In this setting, Ras has been shown to both promote and oppose proliferation and differentiation. By providing control over timing, duration, and signal magnitude, conditional systems allow for more precise investigation of the role of Ras in carcinogenesis, as well as normal cellular physiology. This chapter focuses on use of a ligand-responsive steroid hormone receptor fusion of Ras, ER-Ras, to study aspects of cellular transformation in epidermal keratinocytes.

Animals↗
PubMed2006

Modelling cancer in human skin tissue.

The capacity to induce neoplasia in human tissue in the laboratory has recently provided a new platform for cancer research. Malignant conversion can be achieved in vivo by expressing genes of interest in human tissue that has been regenerated on immune-deficient mice. Induction of cancer in regenerated human skin recapitulates the three-dimensional architecture, tissue polarity, basement membrane structure, extracellular matrix, oncogene signalling and therapeutic target proteins found in intact human skin in vivo. Human-tissue cancer models therefore provide an opportunity to elucidate fundamental cancer mechanisms, to assess the oncogenic potency of mutations associated with specific human cancers and to develop new cancer therapies.

Animals↗
PubMed2005

Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

Animals↗
PubMed2005

Type VII collagen is required for Ras-driven human epidermal tumorigenesis.

Type VII collagen defects cause recessive dystrophic epidermolysis bullosa (RDEB), a blistering skin disorder often accompanied by epidermal cancers. To study the role of collagen VII in these cancers, we examined Ras-driven tumorigenesis in RDEB keratinocytes. Cells devoid of collagen VII did not form tumors in mice, whereas those retaining a specific collagen VII fragment (the amino-terminal noncollagenous domain NC1) were tumorigenic. Forced NC1 expression restored tumorigenicity to collagen VII-null epidermis in a non-cell-autonomous fashion. Fibronectin-like sequences within NC1 (FNC1) promoted tumor cell invasion in a laminin 5-dependent manner and were required for tumorigenesis. Tumor-stroma interactions mediated by collagen VII thus promote neoplasia, and retention of NC1 sequences in a subset of RDEB patients may contribute to their increased susceptibility to squamous cell carcinoma.

Adolescent↗
PubMed2005

CDK4 regulation by TNFR1 and JNK is required for NF-kappaB-mediated epidermal growth control.

Nuclear factor kappaB (NF-kappaB) mediates homeostatic growth inhibition in the epidermis, and a loss of NF-kappaB function promotes proliferation and oncogenesis. To identify mechanisms responsible for these effects, we impaired NF-kappaB action in the epidermis by three different genetic approaches, including conditional NF-kappaB blockade. In each case, epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the G1 cell cycle kinase, CDK4. CDK4 up-regulation required intact TNFR1 and c-Jun NH2-terminal kinase (JNK) function. Cdk4 gene deletion concomitant with conditional NF-kappaB blockade demonstrated that CDK4 is required for growth deregulation. Therefore, epidermal homeostasis depends on antagonist regulation of CDK4 expression by NF-kappaB and TNFR1/JNK.

Animals↗
PubMed2005

Effects of active MEK1 expression in vivo.

Cell transformation is often a result of constitutive activation of genes in signaling pathways that regulate cell proliferation and differentiation. Indeed, the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway is constitutively activated in a large number of cancers. The extent to which a single-gene mutation can alter cell fate, however, remains questionable. In vitro studies have addressed this issue, but organs are comprised of multiple cell types, and in vitro models often poorly approximate these interactions. In response to these limitations, cell-type specific mouse models have been generated as a means to examine the effect of altering a single element of the MAPK pathway in vivo. This review summarizes data from transgenic murine and human tissue models expressing constitutive active forms of MEK1.

Animals↗
PubMed2005

The class II phosphoinositide 3-kinase C2beta is not essential for epidermal differentiation.

Phosphoinositide 3-kinases (PI3Ks) regulate an array of cellular processes and are comprised of three classes. Class I PI3Ks include the well-studied agonist-sensitive p110 isoforms; however, the functions of class II and III PI3Ks are less well characterized. Of the three class II PI3Ks, C2alpha and C2beta are widely expressed in many tissues, including the epidermis, while C2gamma is confined to the liver. In contrast to the class I PI3K p110alpha, which is expressed throughout the epidermis, C2beta was found to be localized in suprabasal cells, suggesting a potential role for C2beta in epidermal differentiation. Overexpressing C2beta in epidermal cells in vitro induced differentiation markers. To study a role for C2beta in tissue, we generated transgenic mice overexpressing C2beta in both suprabasal and basal epidermal layers. These mice lacked epidermal abnormalities. Mice deficient in C2beta were then generated by targeted gene deletion. C2beta knockout mice were viable and fertile and displayed normal epidermal growth, differentiation, barrier function, and wound healing. To exclude compensation by C2alpha, RNA interference was then used to knock down both C2alpha and C2beta in epidermal cells simultaneously. Induction of differentiation markers was unaffected in the absence of C2alpha and C2beta. These findings indicate that class II PI3Ks are not essential for epidermal differentiation.

Animals↗
PubMed2005

Melanoma genetics and the development of rational therapeutics.

Melanoma is a cancer of the neural crest-derived cells that provide pigmentation to skin and other tissues. Over the past 4 decades, the incidence of melanoma has increased more rapidly than that of any other malignancy in the United States. No current treatments substantially enhance patient survival once metastasis has occurred. This review focuses on recent insights into melanoma genetics and new therapeutic approaches being developed based on these advances.

Animals↗
PubMed2004

Mek1 alters epidermal growth and differentiation.

The highly homologous kinases, Mek1 and Mek2, act downstream of Ras and Raf to activate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In epidermis, Ras and Raf promote hyperplasia; however, they act on multiple Mek-independent effectors, and the extent to which Meks can mediate these effects is unknown. To address this, we expressed inducible Meks in transgenic murine and human epidermis. Both Mek1 and Mek2 triggered ERK phosphorylation. Only Mek1, however, recapitulated Ras/Raf effects in increasing proliferation and integrin expression while suppressing differentiation, which are impacts characteristic of epidermal neoplasia. Furthermore, a kinase-dead Mek1 mutant incapable of phosphorylating ERK proteins retained ability to mediate Mek1-driven epidermal proliferation. Mek1 is thus sufficient to promote the proliferative epithelial phenotype in a manner independent of intact kinase function.

Animals↗
PubMed2004

Pathways sufficient to induce epidermal carcinogenesis.

Abnormal epidermal proliferation is characteristic of a number of disorders, including the two most common cancers in the United States, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Both cancers display a disruption in the normal homeostatic balance between cell division and programmed cell death. While abnormal activation of the sonic hedgehog/patched pathway has been established as sufficient to induce hallmark features of BCC in both human and murine epidermis, pathways sufficient to convert normal epidermis into SCC have been less well defined. Building on findings that indicate a potent role for Ras and NF-kappaB in normal epidermal growth regulation, recent work indicates that activation of Ras signaling in concert with inhibition of NF-kappaB function is entirely sufficient to transform normal human epidermis into tumor tissue with all the cardinal features of SCC.

Animals↗
PubMed2004

NF-kappaB RelA opposes epidermal proliferation driven by TNFR1 and JNK.

NF-kappaB inhibition promotes epidermal tumorigenesis; however, whether this reflects an underlying role in homeostasis or a special case confined to neoplasia is unknown. Embryonic lethality of mice lacking NF-kappaB RelA has hindered efforts to address this. We therefore generated developmentally mature RelA(-/-) skin. RelA(-/-) epidermis displays hyperplasia without abnormal differentiation, inflammation, or apoptosis. Hyperproliferation is TNFR1-dependent because Tnfr1 deletion normalized cell division. TNFR1-dependent JNK activation occurred in RelA(-/-) epidermis, and JNK inhibition abolished hyperproliferation due to RelA deficiency. Thus, RelA antagonizes TNFR1-JNK proliferative signals in epidermis and plays a nonredundant role in restraining epidermal growth.

Animals↗
PubMed2004

Targets for molecular therapy of skin cancer.

Cancers of the skin encompass the first and second most common neoplasms in the United States, epidermal basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), respectively, as well as the melanocytic malignancy, malignant melanoma (MM). Recently identified alterations in the function of specific genes in these cancers provide new potential therapeutic targets. These alterations affect conserved regulators of cellular proliferation and viability, including the Sonic Hedgehog, Ras/Raf, ARF/p53, p16(INK4A)/CDK4/Rb and NF-kappaB pathways. New modalities designed to target these specific proteins may represent promising approaches to therapy of human skin cancers.

Animals↗
PubMed2004

Lentivectors for regulated and reversible cutaneous gene delivery.

Systemic administration of therapeutic proteins has value in treating a wide variety of disorders, including erythropoietin (Epo)-responsive anemias. Recombinant proteins, however, are costly and require repeated injections, while gene delivery approaches have suffered from inefficiency and difficulties with regulation. The skin effectively delivers polypeptides to the circulation, and improved approaches would support sustainable, topically regulated protein expression after a single vector injection. Toward this goal, we generated lentivectors in which both gene delivery and persistence in skin are regulated by administration of distinct steroid ligands. Following a single injection of regulated lentivector into human skin regenerated on immunodeficient mice, topical glucocorticoid ligands regulated Epo levels and hematocrit over time. Abrogation of gene delivery was achieved by both glucocorticoid cessation and proviral excision via a 4-hydroxytamoxifen-inducible Cre recombinase. These findings establish an approach to durable, topically controlled systemic delivery of therapeutic proteins from human skin tissue.

Administration, Cutaneous↗
PubMed2004

Intracellular delivery of functional proteins via decoration with transporter peptides.

Despite numerous attractive intracellular targets, protein therapeutics have been principally confined to the extracellular space due to the lack of a straightforward way to deliver functional polypeptides to the cell interior. Peptide sequences facilitating intracellular protein delivery have been identified; however, current strategies to apply them require problematic steps, such as generation of new in-frame fusion proteins, covalent chemical conjugation, and denaturation. We have developed a new approach to protein transfer into cells and tissues that relies on single-step decoration by cysteine-flanked, arginine-rich transporter peptides. This approach facilitated cell and tissue delivery of a variety of functional proteins, including antibodies and enzymes. Decoration with transporter peptides thus provides an attractive general means of intracellular delivery of functional proteins in vitro and in tissue.

Animals↗