Linli Zhou

Perspective of Targeting Cancer-Associated Fibroblasts in Melanoma

Melanoma is known as an exceptionally aggressive and treatment-resistant human cancer. Although a great deal of progress has been made in the past decade, including the development of immunotherapy using immune checkpoint inhibitors and targeted therapy using BRAF, MEK or KIT inhibitors, treatment for unresectable stage III, stage IV, and recurrent melanoma is still challenging with limited response rate, severe side effects and poor prognosis, highlighting an urgent need for discovering and designing more effective approaches to conquer melanoma. Melanoma is not only driven by malignant melanocytes, but also by the altered communication between neoplastic cells and non-malignant cell populations, including fibroblasts, endothelial and inflammatory cells, in the tumor stroma. Infiltrated and surrounding fibroblasts, also known as cancer-associated fibroblasts (CAFs), exhibit both phenotypical and physiological differences compared to normal dermal fibroblasts. They acquire properties of myofibroblasts, remodel the extracellular matrix (ECM) and architecture of the diseased tissue and secrete chemical factors, which all together promote the transformation process by encouraging tumor growth, angiogenesis, inflammation and metastasis and contribute to drug resistance. A number of in vitro and in vivo experiments have shown that stromal fibroblasts promote melanoma cell proliferation and they have been targeted to suppress tumor growth effectively. Evidently, a combination therapy co-targeting tumor cells and stromal fibroblasts may provide promising strategies to improve therapeutic outcomes and overcome treatment resistance. A significant benefit of targeting CAFs is that the approach aims to create a tumor-resistant environment that inhibits growth of melanomas carrying different genetic mutations. However, the origin of CAFs and precise mechanisms by which CAFs contribute to melanoma progression and drug resistance remain poorly understood. In this review, we discuss the origin, activation and heterogeneity of CAFs in the melanoma tumor microenvironment and examine the contributions of stromal fibroblasts at different stages of melanoma development. We also highlight the recent progression in dissecting and characterizing how local fibroblasts become reprogrammed and build a dynamic yet optimal microenvironment for tumors to develop and metastasize. In addition, we review key developments in ongoing preclinical studies and clinical applications targeting CAFs and tumor-stroma interactions for melanoma treatment.

Dermal fibroblasts induce cell cycle arrest and block epithelial–mesenchymal transition to inhibit the early stage melanoma development

Stromal fibroblasts are an integral part of the tumor stroma and constantly interact with cancer cells to promote their initiation and progression. However, the role and function of dermal fibroblasts during the early stage of melanoma development remain poorly understood. We, therefore, designed a novel genetic approach to deactivate stromal fibroblasts at the onset of melanoma formation by targeted ablation of β‐catenin. To our surprise, melanoma tumors formed from β‐catenin‐deficient group (B16F10 mixed with β‐catenin‐deficient fibroblasts) appeared earlier than tumors formed from control group (B16F10 mixed with normal dermal fibroblasts). At the end point when tumors were collected, mutant tumors were bigger and heavier than control tumors. Further analysis showed that there were fewer amounts of stromal fibroblasts and myofibroblasts inside mutant tumor stroma. Melanoma tumors from control group showed reduced proliferation, down‐regulated expression of cyclin D1 and increased expression of cyclin‐dependent kinase inhibitor p16, suggesting dermal fibroblasts blocked the onset of melanoma tumor formation by inducing a cell cycle arrest in B16F10 melanoma cells. Furthermore, we discovered that dermal fibroblasts prevented epithelial‐mesenchymal transition in melanoma cells. Overall, our findings demonstrated that dermal fibroblasts crosstalk with melanoma cells to regulate in vivo tumor development via multiple mechanisms, and the outcomes of their reciprocal interactions depend on activation states of stromal fibroblasts and stages of melanoma development.

Activating β‐catenin signaling in CD133‐positive dermal papilla cells increases hair inductivity

Bioengineering hair follicles using cells isolated from human tissue remains a difficult task. Dermal papilla (DP) cells are known to guide the growth and cycling activities of hair follicles by interacting with keratinocytes. However, DP cells quickly lose their inductivity during in vitro passaging. Rodent DP cell cultures need external addition of growth factors, including WNT and BMP molecules, to maintain the hair inductive property. CD133 is expressed by a subpopulation of DP cells that are capable of inducing hair follicle formation in vivo. We report here that expression of a stabilized form of β‐catenin promoted clonal growth of CD133‐positive (CD133+) DP cells in in vitro three‐dimensional hydrogel culture while maintaining expression of DP markers, including alkaline phosphatase (AP), CD133, and integrin α8. After a 2‐week in vitro culture, cultured CD133+ DP cells with up‐regulated β‐catenin activity led to an accelerated in vivo hair growth in reconstituted skin compared to control cells. Further analysis showed that matrix cell proliferation and differentiation were significantly promoted in hair follicles when β‐catenin signaling was up‐regulated in CD133+ DP cells. Our data highlight an important role for β‐catenin signaling in promoting the inductive capability of CD133+ DP cells for in vitro expansion and in vivo hair follicle regeneration, which could potentially be applied to cultured human DP cells.

YAP and WWTR1: New targets for skin cancer treatment

The core components of the Hippo signaling pathway are a cascade of kinases that govern the phosphorylation of downstream transcriptional co-activators, namely, YES-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ). The Hippo signaling pathway is considered an important tumor-suppressor pathway, and its dysregulation has been noted in a variety of human cancers, in which YAP/WWTR1 enable cancerous cells to overcome contact inhibition, and to grow and spread uncontrollably. Interestingly, however, recent studies have told a somewhat different but perhaps more intriguing YAP/WWTR1 story, as these studies found that YAP/WWTR1 function as a central hub that integrates signals from multiple upstream signaling pathways, cell-cell interactions and mechanical forces and then bind to and activate different downstream transcriptional factors to direct cell social behavior and cell-cell interactions. In this review, we present the latest findings on the role of YAP/WWTR1 in skin physiology, pathology and tumorigenesis and discuss the statuses of newly developed therapeutic interventions that target YAP/WWTR1 in human cancers, as well as their prospects for use as skin cancer treatments.

Activation of β-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth.

The hair follicle dermal papilla (DP) contains a unique prominin-1/CD133-positive (CD133+) cell subpopulation, which has been shown to possess hair follicle-inducing capability. By assaying for endogenous CD133 expression and performing lineage tracing using CD133-CreERT2; ZsGreen1 reporter mice, we find that CD133 is expressed in a subpopulation of DP cells during the growth phase of the murine hair cycle (anagen), but is absent at anagen onset. However, how CD133+ DP cells interact with keratinocytes to induce hair regenerative growth remains unclear. Wnt/β-catenin has long been recognized as a major signaling pathway required for hair follicle morphogenesis, development, and regeneration. Nuclear Wnt/β-catenin activity is observed in the DP during the hair growth phase. Here we show that induced expression of a stabilized form of β-catenin in CD133+ DP cells significantly accelerates spontaneous and depilation-induced hair growth. However, hair follicle regression is not affected in these mutants. Further analysis indicates that CD133+ DP-expressed β-catenin increases proliferation and differentiation of epithelial matrix keratinocytes. Upregulated Wnt/β-catenin activity in CD133+ DP cells also increases the number of proliferating DP cells in each anagen follicle. Our data demonstrate that β-catenin signaling potentiates the capability of CD133+ DP cells to promote postnatal hair growth.

Dermal sheath cells contribute to postnatal hair follicle growth and cycling

Two critical cell types necessary to produce and maintain hair follicles are fibroblasts, which reside in the dermal papilla (DP) and dermal sheath (DS), and keratinocytes, including hair follicle stem cells (HFSCs) and their progeny. Although keratinocytes are the primary constituents of hair follicle that generate the hair structure, the contribution of dermal fibroblasts to hair follicle neogenesis and regeneration is less well defined. Cells within the DP and DS are specialized fibroblasts of mesenchymal origin, collectively forming a dermal microenvironment encircling the epithelial compartment and exhibiting hair cycle-associated plasticity. DP cells (DPCs) of growing follicles are known to possess hair-inducing ability and interact with keratinocytes to achieve regenerative hair growth; however, attempts to elicit human follicle neogenesis with cultured human DPCs have not been very successful partly because they lose inductivity during passaging [1], suggesting that additional efforts are needed to gain a better understanding of the mesenchyme in intact follicles. In particular, the DS, the second dermal compartment, has received rather little attention. The DS is mainly composed of collagen fibers with fibroblasts discontinuously embedded in the middle collagen layer [2]. It has been proposed that cells from the mitotically active DS migrate into the DP at anagen onset, thereby accounting for the significant increase in the total DPC number during anagen [3]. In addition, DSCs close to the DP induced follicle formation when cultured and transplanted into mouse ears and footpads [4]. Rahmani et al. recently beautifully showed that the DS contains a pool of mesenchymal stem cells that can replenish DPCs and DSCs lost during catagen and telogen stages by in vivo lineage tracing using aSMA-CreER transgenic mice [5]. Those findings are not surprising considering the proximity, same embryonic origin and many common biological features between DPCs and DSCs. Cre recombinase under the control of the aSMA promoter in aSMA-CreER transgenic mice is active in vascular and visceral smooth muscle cells as well [6], which may complicate our efforts for hair biology research by utilizing this transgenic mouse strain to recombine specific gene. We, therefore, have introduced an inducible mouse model that expresses Cre recombinase under the control of a fibroblast-specific Col1a2 promoter (Col1a2-CreER) [7], which functions only in active fibroblasts but not in normal static

Natural Antioxidants: Multiple Mechanisms to Protect Skin From Solar Radiation

Human skin exposed to solar ultraviolet radiation (UVR) results in a dramatic increase in the production of reactive oxygen species (ROS). The sudden increase in ROS shifts the natural balance toward a pro-oxidative state, resulting in oxidative stress. The detrimental effects of oxidative stress occur through multiple mechanisms that involve alterations to proteins and lipids, induction of inflammation, immunosuppression, DNA damage, and activation of signaling pathways that affect gene transcription, cell cycle, proliferation, and apoptosis. All of these alterations promote carcinogenesis and therefore, regulation of ROS levels is critical to the maintenance of normal skin homeostasis. Several botanical products have been found to exhibit potent antioxidant capacity and the ability to counteract UV-induced insults to the skin. These natural products exert their beneficial effects through multiple pathways, including some known to be negatively affected by solar UVR. Aging of the skin is also accelerated by UVR exposure, in particular UVA rays that penetrate deep into the epidermis and the dermis where it causes the degradation of collagen and elastin fibers via oxidative stress and activation of matrix metalloproteinases (MMPs). Because natural compounds are capable of attenuating some of the UV-induced aging effects in the skin, increased attention has been generated in the area of cosmetic sciences. The focus of this review is to cover the most prominent phytoproducts with potential to mitigate the deleterious effects of solar UVR and suitability for use in topical application.

Association of TGFβ signaling with the maintenance of a quiescent stem cell niche in human oral mucosa.

A dogma in squamous epithelial biology is that proliferation occurs in the basal cell layer. Notable exceptions are squamous epithelia of the human oral cavity, esophagus, ectocervix, and vagina. In these human epithelia, proliferation is rare in the basal cell layer, and the vast majority of cells positive for Ki67 and other proliferation markers are found in para- and suprabasal cell layers. This unique human feature of a generally quiescent basal cell layer overlaid by highly proliferative cells offers the rare opportunity to study the molecular features of undifferentiated, quiescent, putative stem cells in their natural context. Here, we show that the quiescent human oral mucosa basal cell layer expresses putative markers of stemness, while para- and suprabasal cells are characterized by cell cycle genes. We identified a TGFβ signature in this quiescent basal cell layer. In in vitro organotypic cultures, human keratinocytes could be induced to express markers of these quiescent basal cells when TGFβ signaling is activated. The study suggests that the separation of basal cell layer and proliferation in human oral mucosa may function to accommodate high proliferation rates and the protection of a quiescent reserve stem cell pool. Psoriasis, an epidermal inflammatory hyperproliferative disease, exhibits features of a quiescent basal cell layer mimicking normal oral mucosa. Our data indicate that structural changes in the organization of epithelial proliferation could contribute to longevity and carcinogenesis.

CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth

Active Wnt/β-catenin signaling in the dermal papilla (DP) is required for postnatal hair cycling. In addition, maintenance of the hair-inducing ability of DP cells in vitro requires external addition of Wnt molecules. However, whether DP cells are a critical source of Wnt ligands and induce both autocrine and paracrine signaling cascades to promote adult hair follicle growth and regeneration remains elusive. To address this question, we generated an animal model that allows inducible ablation of Wntless (Wls), a transmembrane Wnt exporter protein, in CD133-positive (CD133+) DP cells. CD133+ cells have been shown to be a specific subpopulation of cells in the DP, which possesses the hair-inducing capability. Here, we show that ablation of Wls expression in CD133+ DP cells results in a shortened period of postnatal hair growth. Mutant hair follicles were unable to enter full anagen (hair growth stage) and progressed toward a rapid regression. Notably, reduced size of the DP and decreased expression of anagen DP marker, versican, were observed in hair follicles when CD133+ DP cells lost Wls expression. Further analysis showed that Wls-deficient CD133+ DP cells led to reduced proliferation and differentiation in matrix keratinocytes and melanocytes that are needed for the generation of the hair follicle structure and a pigmented hair shaft. These findings clearly demonstrate that Wnt ligands produced by CD133+ DP cells play an important role in postnatal hair growth by maintaining the inductivity of DP cells and mediating the signaling cross-talk between the mesenchyme and the epithelial compartment.

Suppression of MAPK signaling in BRAF‐activated PTEN‐deficient melanoma by blocking β‐catenin signaling in cancer‐associated fibroblasts

Cancer‐associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of β‐catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF‐activated PTEN‐deficient mouse melanoma was significantly suppressed in vivo after blocking β‐catenin signaling in CAFs. Further analysis revealed that expression of phospho‐Erk1/2 and phospho‐Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial–mesenchymal transition (EMT)‐like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF‐MEK‐ERK signaling cascade in BRAF‐activated melanoma and may offer a new approach to abrogate host‐dependent drug resistance in targeted therapy.