Association of Squamous Cell Carcinoma and Hyaluronan: A Scope of the Literature.

Background: Nonmelanotic skin cancer (NMSC) refers to cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma. There have been many factors linked with the development of cSCC; however, ultraviolet radiation is the most notable culprit. Mutations in RAS signaling genes, the CDKN2A gene, and genes encoding components of the NOTCH signaling pathways increase the risk of developing cSCC. Many therapeutic approaches are available for cSCC, including chemotherapy, radiation therapy, targeted therapy, immunotherapy, and topical treatment. As cSCC affects millions of people worldwide, there is increasing demand to find more minimally invasive treatment approaches, such as hyaluronic acid therapy. Methods: A narrative literature review was conducted on the available literature regarding NMSC, and various treatment strategies were identified. Conclusions: Recent research investigating whether long-lived cancer-resistant species could yield any potential clues against skin carcinogenesis has highlighted naked mole rats (Heterocephalus glaber). One of the proposed mechanisms associated with this tumor resistance has been the accumulation of high-molecular-weight hyaluronic acid (HMWHA) in the epidermis. Researchers were able to conclude that the CD44/HMWHA interaction mediates cancer cell apoptosis and restricts cell cycle progression as a mechanism of cancer resistance in naked mole rats.

RHAMM regulates MMTV-PyMT-induced lung metastasis by connecting STING-dependent DNA damage sensing to interferon/STAT1 pro-apoptosis signaling.

BACKGROUND: RHAMM is a multifunctional protein that is upregulated in breast tumors, and the presence of strongly RHAMM+ve cancer cell subsets associates with elevated risk of peripheral metastasis. Experimentally, RHAMM impacts cell cycle progression and cell migration. However, the RHAMM functions that contribute to breast cancer metastasis are poorly understood. METHODS: We interrogated the metastatic functions of RHAMM using a loss-of-function approach by crossing the MMTV-PyMT mouse model of breast cancer susceptibility with Rhamm-/- mice. In vitro analyses of known RHAMM functions were performed using primary tumor cell cultures and MMTV-PyMT cell lines. Somatic mutations were identified using a mouse genotyping array. RNA-seq was performed to identify transcriptome changes resulting from Rhamm-loss, and SiRNA and CRISPR/Cas9 gene editing was used to establish cause and effect of survival mechanisms in vitro. RESULTS: Rhamm-loss does not alter initiation or growth of MMTV-PyMT-induced primary tumors but unexpectedly increases lung metastasis. Increased metastatic propensity with Rhamm-loss is not associated with obvious alterations in proliferation, epithelial plasticity, migration, invasion or genomic stability. SNV analyses identify positive selection of Rhamm-/- primary tumor clones that are enriched in lung metastases. Rhamm-/- tumor clones are characterized by an increased ability to survive with ROS-mediated DNA damage, which associates with blunted expression of interferon pathway and target genes, particularly those implicated in DNA damage-resistance. Mechanistic analyses show that ablating RHAMM expression in breast tumor cells by siRNA knockdown or CRISPR-Cas9 gene editing blunts interferon signaling activation by STING agonists and reduces STING agonist-induced apoptosis. The metastasis-specific effect of RHAMM expression-loss is linked to microenvironmental factors unique to tumor-bearing lung tissue, notably high ROS and TGFB levels. These factors promote STING-induced apoptosis of RHAMM+ve tumor cells to a significantly greater extent than RHAMM-ve comparators. As predicted by these results, colony size of Wildtype lung metastases is inversely related to RHAMM expression. CONCLUSION: RHAMM expression-loss blunts STING-IFN signaling, which offers growth advantages under specific microenvironmental conditions of lung tissue. These results provide mechanistic insight into factors controlling clonal survival/expansion of metastatic colonies and has translational potential for RHAMM expression as a marker of sensitivity to interferon therapy.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

RHAMM Is a Multifunctional Protein That Regulates Cancer Progression.

The functional complexity of higher organisms is not easily accounted for by the size of their genomes. Rather, complexity appears to be generated by transcriptional, translational, and post-translational mechanisms and tissue organization that produces a context-dependent response of cells to specific stimuli. One property of gene products that likely increases the ability of cells to respond to stimuli with complexity is the multifunctionality of expressed proteins. Receptor for hyaluronan-mediated motility (RHAMM) is an example of a multifunctional protein that controls differential responses of cells in response-to-injury contexts. Here, we trace its evolution into a sensor-transducer of tissue injury signals in higher organisms through the detection of hyaluronan (HA) that accumulates in injured microenvironments. Our goal is to highlight the domain and isoform structures that generate RHAMM's function complexity and model approaches for targeting its key functions to control cancer progression.

Function-Blocking RHAMM Peptides Attenuate Fibrosis and Promote Antifibrotic Adipokines in a Bleomycin-Induced Murine Model of Systemic Sclerosis.

Systemic sclerosis a chronic, fibrotic disorder associated with high disease-specific mortality and morbidity. Cutaneous manifestations include dermal thickening and obliteration of dermal adipose tissue. Accumulation of low-molecular-weight hyaluronan, which signals through the receptor for hyaluronan-mediated motility, RHAMM, leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. The purpose of this study is to test the efficacy of two function-blocking RHAMM peptides, NPI-110 and NPI-106, in reducing skin fibrosis in a bleomycin-induced mouse model of systemic sclerosis. NPI-110 reduced visible measures of fibrosis (dermal thickness and collagen production, deposition, and organization) and profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1). NPI-110 treatment also increased the expression of the antifibrotic adipokines perilipin and adiponectin. Both RHAMM peptides strongly reduced dermal RHAMM expression, predicting that dermal fibroblasts are peptide targets. Transcriptome and cell culture analyses using Rhamm-/- and Rhamm-rescued dermal fibroblasts reveal a TGFß1/RHAMM/MYC signaling axis that promotes fibrogenic gene expression and myofibroblast differentiation. RHAMM function‒blocking peptides suppress this signaling and prevent TGFß1-induced myofibroblast differentiation. These results suggest that inhibiting RHAMM signaling will offer a treatment method for cutaneous fibrosis in systemic sclerosis.

Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context-dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor-regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

A Hyaluronan-binding Peptide (P15-1) Reduces Inflammatory and Catabolic Events in IL-1ß-treated Human Articular Chondrocytes.

Inflammation plays a critical role in osteoarthritis (OA). It stimulates catabolic events in articular chondrocytes and prevents chondrogenic precursor cells from repairing cartilage lesions, leading to accelerated cartilage degradation. Therefore, the identification of novel factors that reduce catabolic events in chondrocytes and enhances chondrogenic differentiation of precursor cells in an inflammatory environment may provide novel therapeutic strategies for the treatment of OA. The goal of this study was to determine whether a hyaluronan (HA)-binding peptide (P15-1), via interacting with high molecular weight (HMW)HA can enhance the anti-inflammatory properties of HMWHA and decrease catabolic events in interleukin-1beta (IL-1ß)-treated human articular chondrocytes. Treatment with P15-1 decreased catabolic events and stimulated anabolic events in articular chondrocytes cultured in an inflammatory environment. P15-1 pre-mixed with HMWHA was more effective in inhibiting catabolic events and stimulating anabolic events than P15-1 or HMWHA alone. Our findings suggest that P15-1 together with HMWHA inhibits catabolic events in articular chondrocytes via the inhibition of p38 mitogen-activated protein kinases (MAPK) and increasing the thickness of the pericellular matrix (PCM) around chondrocytes thereby decreasing catabolic signaling. Finally, conditioned medium from IL-1ß and P15-1-treated human articular chondrocytes was less inhibitory for chondrogenic differentiation of precursor cells than conditioned medium from chondrocytes treated with IL-1ß alone. In conclusion, P15-1 is proposed to function synergistically with HMWHA to enhance the protective microenvironment for chondrocytes and mesenchymal stem cells during inflammation and regeneration.

Creating a Favorable Microenvironment for Fat Grafting in a Novel Model of Radiation-Induced Mammary Fat Pad Fibrosis.

BACKGROUND: Radiofibrosis of breast tissue compromises breast reconstruction by interfering with tissue viability and healing. Autologous fat transfer may reduce radiotherapy-related tissue injury, but graft survival is compromised by the fibrotic microenvironment. Elevated expression of receptor for hyaluronan-mediated motility (RHAMM; also known as hyaluronan-mediated motility receptor, or HMMR) in wounds decreases adipogenesis and increases fibrosis. The authors therefore developed RHAMM peptide mimetics to block RHAMM profibrotic signaling following radiation. They propose that this blocking peptide will decrease radiofibrosis and establish a microenvironment favoring adipose-derived stem cell survival using a rat mammary fat pad model. METHODS: Rat mammary fat pads underwent a one-time radiation dose of 26 Gy. Irradiated (n = 10) and nonirradiated (n = 10) fat pads received a single intramammary injection of a sham injection or peptide NPI-110. Skin changes were examined clinically. Mammary fat pad tissue was processed for fibrotic and adipogenic markers using quantitative polymerase chain reaction and immunohistochemical analysis. RESULTS: Clinical assessments and molecular analysis confirmed radiation-induced acute skin changes and radiation-induced fibrosis in rat mammary fat pads. Peptide treatment reduced fibrosis, as detected by polarized microscopy of picrosirius red staining, increased collagen ratio of 3:1, reduced expression of collagen-1 crosslinking enzymes lysyl-oxidase, transglutaminase 2, and transforming growth factor ß1 protein, and increased adiponectin, an antifibrotic adipokine. RHAMM was expressed in stromal cell subsets and was downregulated by the RHAMM peptide mimetic. CONCLUSION: Results from this study predict that blocking RHAMM function in stromal cell subsets can provide a postradiotherapy microenvironment more suitable for fat grafting and breast reconstruction.

Chondroitin sulfate proteoglycan 4 enhanced melanoma motility and growth requires a cysteine in the core protein transmembrane domain.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell surface proteoglycan that enhances malignant potential in melanoma and several other tumor types. CSPG4 functions as a transmembrane scaffold in melanoma cells to activate oncogenic signaling pathways such as focal adhesion kinase (FAK) and extracellular signal regulated kinases 1,2, that control motility, invasion and anchorage independent growth. Here, we demonstrate that CSPG4 promotes directional motility and anchorage independent growth of melanoma cells by organizing and positioning a signaling complex containing activated FAK to lipid rafts within the plasma membrane of migrating cells. This FAK-containing signal transduction platform, which consists of syntenin-1, active Src and caveolin-1 requires the cytoplasmic domain of CSPG4 for assembly. Enhanced directional motility promoted by this complex also requires a CSPG4 transmembrane cysteine residue C2230. Substituting C2230 with alanine (CSPG4) still permits assembly of the signaling complex, however Src remains in an inactive state. CSPG4 also fails to promote anchorage independent growth and activation of extracellular signal regulated kinases 1,2. Therapies that target the transmembrane domain of CSPG4 could be a novel strategy for limiting progression by disrupting its function as a compartmentalized motogenic and growth-promoting oncogenic signaling node.

Corrigendum: Hyaluronan, Cancer-Associated Fibroblasts and the Tumor Microenvironment in Malignant Progression.

[This corrects the article DOI: 10.3389/fcell.2018.00048.].

Spatial statistical tools for genome-wide mutation cluster detection under a microarray probe sampling system.

Mutation cluster analysis is critical for understanding certain mutational mechanisms relevant to genetic disease, diversity, and evolution. Yet, whole genome sequencing for detection of mutation clusters is prohibitive with high cost for most organisms and population surveys. Single nucleotide polymorphism (SNP) genotyping arrays, like the Mouse Diversity Genotyping Array, offer an alternative low-cost, screening for mutations at hundreds of thousands of loci across the genome using experimental designs that permit capture of de novo mutations in any tissue. Formal statistical tools for genome-wide detection of mutation clusters under a microarray probe sampling system are yet to be established. A challenge in the development of statistical methods is that microarray detection of mutation clusters is constrained to select SNP loci captured by probes on the array. This paper develops a Monte Carlo framework for cluster testing and assesses test statistics for capturing potential deviations from spatial randomness which are motivated by, and incorporate, the array design. While null distributions of the test statistics are established under spatial randomness via the homogeneous Poisson process, power performance of the test statistics is evaluated under postulated types of Neyman-Scott clustering processes through Monte Carlo simulation. A new statistic is developed and recommended as a screening tool for mutation cluster detection. The statistic is demonstrated to be excellent in terms of its robustness and power performance, and useful for cluster analysis in settings of missing data. The test statistic can also be generalized to any one dimensional system where every site is observed, such as DNA sequencing data. The paper illustrates how the informal graphical tools for detecting clusters may be misleading. The statistic is used for finding clusters of putative SNP differences in a mixture of different mouse genetic backgrounds and clusters of de novo SNP differences arising between tissues with development and carcinogenesis.

A truncated RHAMM protein for discovering novel therapeutic peptides.

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706-767), 7 kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7 kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7 kDa RHAMM. Therefore, in terms of its key binding properties, the 7 kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.

Hyaluronan, Cancer-Associated Fibroblasts and the Tumor Microenvironment in Malignant Progression.

This review summarizes the roles of CAFs in forming a "cancerized" fibrotic stroma favorable to tumor initiation and dissemination, in particular highlighting the functions of the extracellular matrix component hyaluronan (HA) in these processes. The structural complexity of the tumor and its host microenvironment is now well appreciated to be an important contributing factor to malignant progression and resistance-to-therapy. There are multiple components of this complexity, which include an extensive remodeling of the extracellular matrix (ECM) and associated biomechanical changes in tumor stroma. Tumor stroma is often fibrotic and rich in fibrillar type I collagen and hyaluronan (HA). Cancer-associated fibroblasts (CAFs) are a major source of this fibrotic ECM. CAFs organize collagen fibrils and these biomechanical alterations provide highways for invading carcinoma cells either under the guidance of CAFs or following their epithelial to mesenchymal transition (EMT). The increased HA metabolism of a tumor microenvironment instructs carcinoma initiation and dissemination by performing multiple functions. The key effects of HA reviewed here are its role in activating CAFs in pre-malignant and malignant stroma, and facilitating invasion by promoting motility of both CAFs and tumor cells, thus facilitating their invasion. Circulating CAFs (cCAFs) also form heterotypic clusters with circulating tumor cells (CTC), which are considered to be pre-cursors of metastatic colonies. cCAFs are likely required for extravasation of tumors cells and to form a metastatic niche suitable for new tumor colony growth. Therapeutic interventions designed to target both HA and CAFs in order to limit tumor spread and increase response to current therapies are discussed.

Hyaluronan Isolation from Mouse Mammary Gland.

The glycosaminoglycan hyaluronan (HA) is a key component of the extracellular matrix. The molecular weight of HA is heterogeneous and can reach from several million to several hundred daltons. The effect of HA on cell behavior is size dependent; fragmented HA acts as a danger signal, stimulates cell migration and proliferation and is proinflammatory, native high molecular weight HA suppresses inflammation. Therefore, it is important to analyze HA size distribution when studying the role of HA in tissue homeostasis and pathology. This protocol describes isolation of HA from mouse mammary glands but can also be applied to other tissues. The quality of the isolated HA is sufficient to analyze size distribution by gel electrophoresis ( Calabro et al., 2000 ).

Mouse Mammary Gland Whole Mount Preparation and Analysis.

The mammary gland undergoes extensive remodeling during pregnancy and is also subject to neoplastic processes both of which result in histological changes of the gland epithelial structure. Since the mammary tree is a complex three-dimensional structure a method is needed that provides an overview of the entire gland. Whole mounts provide this information, are inexpensive and do not require specialized equipment. This protocol describes mammary gland isolation, whole mount preparation and analysis. Mammary gland tissue, which is removed postmortem, is stained with Carmine Alum, a nuclear stain, allowing detection of epithelial structures embedded in the adipose tissue of the mammary fat pad. Stained mammary glands are imaged by light microscopy or embedded and sectioned for histological examination. Image analysis software such as Image J can be used to quantify extensity of branching complexity, epithelial structure remodeling or hyperplastic changes.

Hyaluronan modulates growth factor induced mammary gland branching in a size dependent manner.

Mammary gland morphogenesis begins during fetal development but expansion of the mammary tree occurs postnatally in response to hormones, growth factors and extracellular matrix. Hyaluronan (HA) is an extracellular matrix polysaccharide that has been shown to modulate growth factor-induced branching in culture. Neither the physiological relevance of HA to mammary gland morphogenesis nor the role that HA receptors play in these responses are currently well understood. We show that HA synthase (HAS2) is expressed in both ductal epithelia and stromal cells but HA primarily accumulates in the stroma. HA accumulation and expression of the HA receptors CD44 and RHAMM are highest during gestation when gland remodeling, lateral branch infilling and lobulo-alveoli formation is active. Molecular weight analyses show that approximately 98% of HA at all stages of morphogenesis is >300kDa. Low levels of 7-114kDa HA fragments are also detected and in particular the accumulation of 7-21kDa HA fragments are significantly higher during gestation than other morphogenetic stages (p<0.05). Using these in vivo results as a guide, in culture analyses of mammary epithelial cell lines (EpH4 and NMuMG) were performed to determine the roles of high molecular weight, 7-21kDa (10kDa MWavg) and HA receptors in EGF-induced branching morphogenesis. Results of these assays show that while HA synthesis is required for branching and 10kDa HA fragments strongly stimulate branching, the activity of HA decreases with increasing molecular weight and 500kDa HA strongly inhibits this morphogenetic process. The response to 10kDa HA requires RHAMM function and genetic deletion of RHAMM transiently blunts lateral branching in vivo. Collectively, these results reveal distinct roles for HA polymer size in modulating growth factor induced mammary gland branching and implicates these polymers in both the expansion and sculpting of the mammary tree during gestation.

Biphasic Dependence of Glioma Survival and Cell Migration on CD44 Expression Level.

While several studies link the cell-surface marker CD44 to cancer progression, conflicting results show both positive and negative correlations with increased CD44 levels. Here, we demonstrate that the survival outcomes of genetically induced glioma-bearing mice and of high-grade human glioma patients are biphasically correlated with CD44 level, with the poorest outcomes occurring at intermediate levels. Furthermore, the high-CD44-expressing mesenchymal subtype exhibited a positive trend of survival with increased CD44 level. Mouse cell migration rates in ex vivo brain slice cultures were also biphasically associated with CD44 level, with maximal migration corresponding to minimal survival. Cell simulations suggest that cell-substrate adhesiveness is sufficient to explain this biphasic migration. More generally, these results highlight the potential importance of non-monotonic relationships between survival and biomarkers associated with cancer progression.