Up-regulation of Histone Methyltransferase, DOT1L, by Matrix Hyaluronan Promotes MicroRNA-10 Expression Leading to Tumor Cell Invasion and Chemoresistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma.

Human head and neck squamous cell carcinoma is a solid tumor malignancy associated with major morbidity and mortality. In this study, we determined that human head and neck squamous cell carcinoma-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by a high level of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. Importantly, matrix hyaluronan (HA) induces the up-regulation of stem cell markers that display the hallmark CSC properties. Histone methyltransferase, DOT1L, is also up-regulated by HA in CSCs (isolated from HSC-3 cells). Further analyses indicate that the stimulation of microRNA-10b (miR-10b) expression is DOT1L-specific and HA/CD44-dependent in CSCs. This process subsequently results in the overexpression of RhoGTPases and survival proteins leading to tumor cell invasion and cisplatin resistance. Treatment of CSCs with DOT1L-specific small interfering RNAs (siRNAs) effectively blocks HA/CD44-mediated expression of DOT1L, miR-10b production, and RhoGTPase/survival protein up-regulation as well as reduces tumor cell invasion and enhances chemosensitivity. CSCs were also transfected with a specific anti-miR-10b inhibitor to silence miR-10b expression and block its target functions. Our results demonstrate that the anti-miR-10 inhibitor not only decreases RhoGTPase/survival protein expression and tumor cell invasion, but also increases chemosensitivity in HA-treated CSCs. Taken together, these findings strongly support the contention that histone methyltransferase, DOT1L-associated epigenetic changes induced by HA play pivotal roles in miR-10 production leading to up-regulation of RhoGTPase and survival proteins. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, tumor cell invasion, and chemotherapy resistance in HA/CD44-activated head and neck cancer.

Activation of Matrix Hyaluronan-Mediated CD44 Signaling, Epigenetic Regulation and Chemoresistance in Head and Neck Cancer Stem Cells.

Head and neck squamous cell carcinoma (HNSCC) is a solid tumor composed by a genotypically and phenotypically heterogeneous population of neoplastic cells types. High recurrence rate and regional metastases lead to major morbidity and mortality. Recently, many studies have focused on cellular and molecular mechanisms of tumor progression that can help to predict prognosis and to choose the best therapeutic approach for HNSCC patients. Hyaluronan (HA), an important glycosaminoglycan component of the extracellular matrix (ECM), and its major cell surface receptor, CD44, have been suggested to be important cellular mediators influencing tumor progression and treatment resistance in head and neck cancer. HNSCC contains a small subpopulation of cells that exhibit a hallmark of CD44-expressing cancer stem cell (CSC) properties with self-renewal, multipotency, and a unique potential for tumor initiation. HA has been shown to stimulate a variety of CSC functions including self-renewal, clone formation and differentiation. This review article will present current evidence for the existence of a unique small population of CD44v3highALDHhigh-expressing CSCs in HNSCC. A special focus will be placed on the role of HA/CD44-induced oncogenic signaling and histone methyltransferase, DOT1L activities in regulating histone modifications (via epigenetic changes) and miRNA activation. Many of these events are essential for the CSC properties such as Nanog/Oct4/Sox2 expression, spheroid/clone formation, self-renewal, tumor cell migration/invasion, survival and chemotherapeutic drug resistance in HA-activated head and neck cancer. These newly-discovered HA/CD44-mediated oncogenic signaling pathways delineate unique tumor dynamics with implications for defining the drivers of HNSCC progression processes. Most importantly, the important knowledge obtained from HA/CD44-regulated CSC signaling and functional activation could provide new information regarding the design of novel drug targets to overcome current therapeutic drug resistance which will have significant treatment implications for head and neck cancer patients.

Matrix Hyaluronan Promotes Specific MicroRNA Upregulation Leading to Drug Resistance and Tumor Progression.

Solid tumor invasion, metastasis and therapeutic drug resistance are the common causes for serious morbidity and cancer recurrence in patients. A number of research studies have searched for malignancy-related biomarkers and drug targets that are closely linked to tumor cell properties. One of the candidates is matrix hyaluronan (HA), which is known as one of the major extracellular matrix (ECM) components. HA serves as a physiological ligand for surface CD44 molecule and also functions as a bio-regulator. The binding of HA to CD44 has been shown to stimulate concomitant activation of a number of oncogenic pathways and abnormal cellular processes in cancer cells and cancer stem cells (CSCs). MicroRNAs (miRNAs) belong to a class of small RNAs containing ~20-25 nucleotides and are known to promote aberrant cellular functions in cancer cells. In this article, I have focused on the role of HA interaction with CD44 and several important signaling molecules in the regulation of unique miRNAs (e.g., miR-21, miR-302 and miR-10b) and their downstream targets leading to multiple tumor cell-specific functions (e.g., tumor cell growth, drug resistance and metastasis) and cancer progression. This new knowledge could provide the groundwork necessary for establishing new tumor markers and developing important, novel drugs targeted against HA/CD44-associated tumor progression, which can be utilized in the therapeutic treatment of metastatic cancer patients.

Matrix hyaluronan-activated CD44 signaling promotes keratinocyte activities and improves abnormal epidermal functions.

Hyaluronan (HA), a major component of the extracellular matrix, is enriched in skin tissues, particularly the epidermis. HA binds to a ubiquitous, abundant, and functionally important family of cell surface receptors, CD44. This article reviews the current evidence for HA/CD44-mediated activation of RhoGTPase signaling and calcium mobilization, leading to the regulation of keratinocyte activities and various epidermal functions. It further discusses the role of HA-mediated CD44 interactions with unique downstream effectors, such as RhoGTPases (RhoA and Rac1), Rho-kinase, protein kinase-Nγ, and phosphoinositide-specific phospholipases (phospholipases Cε and Cγ1) in coordinating certain intracellular signaling pathways, such as calcium mobilization, phosphatidylinositol 3-kinase-AKT activation, cortactin-actin binding, and actin-associated cytoskeleton reorganization; generating the onset of important keratinocyte activities, such as cell adhesion, proliferation, migration, and differentiation; and performing epidermal functions. Topical application of selective HA fragments (large versus small HA) to the skin of wild-type mice (but not CD44 knockout mice) improves keratinocyte-associated epidermal functions and accelerates permeability barrier recovery and skin wound healing. Consequently, specific HA fragment (large versus small HA)-mediated signaling events (through the CD44 receptor) are required for keratinocyte activities, which offer new HA-based therapeutic options for patients experiencing epidermal dysfunction and skin damage as well as aging-related skin diseases, such as epidermal thinning (atrophy), permeability barrier dysfunction, and chronic nonhealing wounds.

Identification of novel drugs to target dormant micrometastases.

BACKGROUND: Cancer-specific survival has changed remarkably little over the past half century, mainly because metastases that are occult at diagnosis and generally resistant to chemotherapy subsequently develop months, years or even decades following definitive therapy. Targeting the dormant micrometastases responsible for these delayed or occult metastases would represent a major new tool in cancer patient management. Our hypothesis is that these metastases develop from micrometastatic cells that are suppressed by normal extracellular matrix (ECM). METHODS: A new screening method was developed that compared the effect of drugs on the proliferation of cells grown on a normal ECM gel (small intestine submucosa, SISgel) to cells grown on plastic cell culture plates. The desired endpoint was that cells on SISgel were more sensitive than the same cells grown as monolayers. Known cancer chemotherapeutic agents show the opposite pattern. RESULTS: Screening 13,000 compounds identified two leads with low toxicity in mice and EC50 values in the range of 3-30 µM, depending on the cell line, and another two leads that were too toxic to mice to be useful. In a novel flank xenograft method of suppressed/dormant cells co-injected with SISgel into the flank, the lead compounds significantly eliminated the suppressed cells, whereas conventional chemotherapeutics were ineffective. Using a 4T1 triple negative breast cancer model, modified for physiological metastatic progression, as predicted, both lead compounds reduced the number of large micrometastases/macrometastases in the lung. One of the compounds also targeted cancer stem cells (CSC) isolated from the parental line. The CSC also retained their stemness on SISgel. Mechanistic studies showed a mild, late apoptotic response and depending on the compound, a mild arrest either at S or G2/M in the cell cycle. CONCLUSIONS: In summary we describe a novel, first in class set of compounds that target micrometastatic cells and prevent their reactivation to form recurrent tumors/macrometastases.

Hyaluronan-CD44 interaction promotes c-Jun signaling and miRNA21 expression leading to Bcl-2 expression and chemoresistance in breast cancer cells.

MicroRNA-21 (miR-21) is associated with the development of solid tumors progression including breast cancer. In this study we investigated matrix hyaluronan (HA)-CD44 (a primary HA receptor) interaction with c-Jun N-Terminal Kinase (JNK)/c-Jun signaling in MDA-MB-468 breast cancer cells [a triple-negative (estrogen receptor-negative/progesterone receptor-negative/HER2-negative) breast cancer cell line]. Our results indicated that HA binding to CD44 promotes c-Jun nuclear translocation and transcriptional activation. Further analyses revealed that miR-21 is regulated by an upstream promoter containing AP1 binding site(s), and chromatin immunoprecipitation (CHIP) assays demonstrated that stimulation of miR-21 expression by HA/CD44 interaction is c-Jun-dependent in these breast cancer cells. This process results in an increase of the anti-apoptosis protein Bcl-2 and upregulation of inhibitors of the apoptosis family of proteins (IAPs) as well as chemoresistance in MDA-MB-468 cells. Treatment with c-Jun specific small interfering RNAs effectively blocks HA-mediated c-Jun signaling and abrogates miR-21 production as well as causes downregulation of survival proteins (Bcl-2 and IAPs) and enhancement of chemosensitivity. In addition, our results demonstrated that anti-miR-21 inhibitor not only downregulates Bcl-2/IAP expression but also increases chemosensitivity in HA-treated breast cancer cells. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in miR-21 production leading to survival protein (Bcl-2/IAP) upregulation and chemoresistance in triple negative breast cancer cells such as MDA-MB-468 cell line. This novel HA/CD44-mediated c-Jun signaling pathway and miR-21 production provide a new drug target for the future intervention strategies to treat breast cancer.

The inhibition of miR-21 promotes apoptosis and chemosensitivity in ovarian cancer.

BACKGROUND: MicroRNAs have been implicated in tumorigenesis, drug resistance, and prognosis in cancer. We investigated the role of microRNA-21 (miR-21) in regulating ovarian cancer drug resistance. METHODS: We used parental and cisplatin resistant ovarian cell lines to demonstrate the role of miR-21 in drug resistance and investigated the gene targets of miR-21. Fresh tumor specimens were used to validate our in vitro findings. RESULTS: Cisplatin resistant ovarian cells were four-fold more resistant compared to the parental cell line. MiR-21 was overexpressed in the resistant cell line on microRNA microarray, which was subsequently validated with qRT-PCR. Using anti-microRNA inhibitors, we demonstrated that miR-21 attenuation reversed the drug resistant phenotype in both the resistant and parental cell lines. The inhibition of miR-21 induced apoptosis based on annexin V-FITC immunostaining. Using Western blot analysis, miR-21 knockdown enhanced the expression of tumor suppressor PDCD4, and attenuated apoptosis inhibitor c-IAP2. Using 101 specimens from advanced ovarian cancer patients enrolled in The Cancer Genome Atlas, we found that women with tumors that overexpressed miR-21 were associated with a shorter progression-free survival. CONCLUSION: Our data suggest that miR-21 regulates drug resistance via apoptosis and cellular survival pathways. Targeting miR-21 may have clinical utility in the treatment of resistant ovarian cancer.

Hyaluronan-CD44v3 interaction with Oct4-Sox2-Nanog promotes miR-302 expression leading to self-renewal, clonal formation, and cisplatin resistance in cancer stem cells from head and neck squamous cell carcinoma.

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Role of hyaluronan-mediated CD44 signaling in head and neck squamous cell carcinoma progression and chemoresistance.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Hyaluronan-CD44 interaction promotes c-Src-mediated twist signaling, microRNA-10b expression, and RhoA/RhoC up-regulation, leading to Rho-kinase-associated cytoskeleton activation and breast tumor cell invasion.

Dysregulation of microRNAs is observed in many cancers, including breast cancer. In particular, miR-10b appears to play an important role in tumor cell invasion and breast cancer progression. In this study, we investigated hyaluronan (HA)-induced CD44 (a primary HA receptor) interaction with c-Src kinase and the transcriptional factor, Twist, in breast tumor cells (MDA-MB-231 cells). Our results indicate that HA binding to CD44 promotes c-Src kinase activation, which, in turn, increases Twist phosphorylation, leading to the nuclear translocation of Twist and transcriptional activation. Further analyses reveal that miR-10b is controlled by an upstream promoter containing the Twist binding site(s), whereas ChIP assays demonstrate that stimulation of miR-10b expression by HA/CD44-activated c-Src is Twist-dependent in breast tumor cells. This process results in the reduction of a tumor suppressor protein (HOXD10), RhoA/RhoC up-regulation, Rho-kinase (ROK) activation, and breast tumor cell invasion. Treatment of MDA-MB-231 cells with PP2 (a c-Src inhibitor) or Twist-specific siRNAs effectively blocks HA-mediated Twist signaling events, abrogates miR-10b production, and increases HOXD10 expression. Subsequently, this c-Src/Twist signaling inhibition causes down-regulation of RhoA/RhoC expression and impairment of ROK-regulated cytoskeleton function (e.g. tumor cell invasion). To further evaluate the role of miR-10b in RhoGTPase signaling, MDA-MB-231 cells were also transfected with a specific anti-miR-10b inhibitor in order to silence miR-10b expression and block its target functions. Our results demonstrate that anti-miR-10b inhibitor not only enhances HOXD10 expression but also abrogates HA/CD44-mediated tumor cell behaviors in breast tumor cells. Taken together, these findings indicate that the HA-induced CD44 interaction with c-Src-activated Twist plays a pivotal role in miR-10b production, leading to the down-regulation of tumor suppressor protein (HOXD10), RhoGTPase-ROK activation, and tumor cell invasion. All of these events are critical prerequisite steps for the acquisition of metastatic properties by human breast cancer cells.

Hyaluronan-CD44 interaction with protein kinase C(epsilon) promotes oncogenic signaling by the stem cell marker Nanog and the Production of microRNA-21, leading to down-regulation of the tumor suppressor protein PDCD4, anti-apoptosis, and chemotherapy resistance in breast tumor cells.

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression.

Hyaluronan (HA), a major component of the extracellular matrix (ECM), is enriched in many types of tumors. In cancer patients HA concentrations are usually higher in malignant tumors than in corresponding benign or normal tissues, and in some tumor types the level of HA is predictive of malignancy. HA is often bound to CD44 isoforms which are ubiquitous, abundant, and functionally important cell surface receptors. This article reviews the current evidence for HA/CD44-mediated activation of the ankyrin-based cytoskeleton and RhoGTPase signaling during tumor progression. A special focus is placed on the role of HA-mediated CD44 interaction with unique downstream effectors (e.g., the cytoskeletal protein, ankyrin and/or various GTPases (e.g., RhoA, Rac1 and Cdc42)) in coordinating intracellular signaling pathways (e.g., Ca(2+) mobilization, Rho signaling, PI3 kinase-AKT activation, NHE1-mediated cellular acidification, transcriptional upregulation and cytoskeletal function) and generating the concomitant onset of tumor cell activities (e.g., tumor cell adhesion, growth, survival, migration and invasion) and tumor progression. I believe this information will provide valuable new insights into poorly understood aspects of solid tumor malignancy. Furthermore, the new knowledge concerning HA/CD44-mediated oncogenic signaling events will have potentially important clinical utility, and could establish CD44 and its associated signaling molecules as important tumor markers for the early detection and evaluation of oncogenic potential. It could also serve as ground work for the future development of new drug targets to inhibit HA/CD44-mediated tumor metastasis and cancer progression.

Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord.

In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine whether MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.

The role of CD44 in cutaneous inflammation.

CD44 is a transmembrane glycoprotein expressed in various tissues including the skin. Previous studies indicated that CD44 is required for epidermal permeability barrier homeostasis and keratinocyte differentiation. Yet, while some studies have demonstrated that CD44 is critical for the development of inflammation, others have shown that CD44 is not essential for the development of cutaneous inflammation. In this study, we evaluated the changes in epidermal CD44 expression in a variety of skin inflammatory models and determined whether CD44 is required for the development of cutaneous inflammation. Inflammatory responses were compared in CD44 KO versus wild-type mice in acute models of irritant and allergic contact dermatitis, as well as in a subacute allergic contact dermatitis induced by repeated hapten treatment. Inflammatory responses were assessed by measuring ear thickness and epidermal hyperplasia in haematoxylin & eosin-stained sections. Our results demonstrate that: (i) epidermal CD44 expression increases in both acute and subacute cutaneous inflammatory models; and (ii) acute disruption of the epidermal permeability barrier function increases epidermal CD44 expression. Whereas inflammatory responses did not differ between CD44 KO and wild-type mice in acute models of irritant and allergic contact dermatitis, both inflammatory responses and epidermal hyperplasia increased in CD44 KO mice following repeated hapten challenges. These results show first, that permeability barrier disruption and inflammation stimulate epidermal CD44 expression, and second, that CD44 modulates epidermal proliferation and inflammatory responses in a subacute murine allergic contact dermatitis model.

Matrix Hyaluronan-CD44 Interaction Activates MicroRNA and LncRNA Signaling Associated With Chemoresistance, Invasion, and Tumor Progression.

Tumor malignancies involve cancer cell growth, issue invasion, metastasis and often drug resistance. A great deal of effort has been placed on searching for unique molecule(s) overexpressed in cancer cells that correlate(s) with tumor cell-specific behaviors. Hyaluronan (HA), one of the major ECM (extracellular matrix) components have been identified as a physiological ligand for surface CD44 isoforms which are frequently overexpressed in malignant tumor cells during cancer progression. The binding interaction between HA and CD44 isoforms often stimulates aberrant cellular signaling processes and appears to be responsible for the induction of multiple oncogenic events required for cancer-specific phenotypes and behaviors. In recent years, both microRNAs (miRNAs) (with ~20-25 nucleotides) and long non-coding RNAs (lncRNAs) (with ~200 nucleotides) have been found to be abnormally expressed in cancer cells and actively participate in numerous oncogenic signaling events needed for tumor cell-specific functions. In this review, I plan to place a special emphasis on HA/CD44-induced signaling pathways and the presence of several novel miRNAs (e.g., miR-10b/miR-302/miR-21) and lncRNAs (e.g., UCA1) together with their target functions (e.g., tumor cell migration, invasion, and chemoresistance) during cancer development and progression. I believe that important information can be obtained from these studies on HA/CD44-activated miRNAs and lncRNA that may be very valuable for the future development of innovative therapeutic drugs for the treatment of matrix HA/CD44-mediated cancers.

Hyaluronan-CD44 interaction promotes HPV 16 E6 oncogene-mediated oropharyngeal cell carcinoma survival and chemoresistance.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that often involves the oral cavity, pharynx, larynx, or paranasal sinuses. There is a compelling evidence of the human papilloma virus including HPV16 E6 oncogene drives cell transformation and oncogenic processes of HPV positive (HVP+) HNSCC [in particular, Oropharyngeal Squamous Cell Carcinoma (OPSCC)]. In this study, we determined that human OPSCC-derived, HPV16 E6+ cells (UMSCC-104 and UMSCC-47 cell lines) express CD44 and a regulatory transcription factor, c-Jun. Importantly, interaction between matrix hyaluronan (HA) and CD44 (an HA receptor) promotes c-Jun phosphorylation followed by phospho-c-Jun nuclear translocation and co-localization with HPV16 E6 in the nucleus of both UMSCC-104 and UMSCC-47 cells. Further analyses revealed that HPV16 E6 expression is regulated by an upstream promoter containing AP1/c-Jun binding site(s), and chromatin immunoprecipitation (ChIP) assays demonstrated that stimulation of HPV16 E6 expression by HA-CD44 interaction is phospho-c-Jun dependent in these HPV16+ UMSCC-104 and UMSCC-47 cells. This process results in an upregulation of survival proteins, inhibitors of the apoptosis family of proteins (IAPs) and chemoresistance in these HPV16+ cells. Treatment of UMSCC-104 or UMSCC-47 cells with c-Jun-specific or HPV16 E6-specific small interfering RNAs effectively blocks HA/CD44-mediated c-Jun signaling and abrogates HPV16 E6 expression as well as causes downregulation of survival proteins (cIAP-1 and cIAP-2) expression and enhancement of chemosensitivity. Together, these findings suggest that the HA/CD44-induced c-Jun signaling plays a pivotal role in HPV16 E6 upregulation leading to survival protein (cIAP-1/cIAP-2) production and chemoresistance in HPV16+ UMSCC-104 and UMSCC-47 cells. Most importantly, using a mouse xenograft model, we have observed that Cisplatin chemotherapy combined with the suppression of CD44, c-Jun and HPV16 E6 (by treating both UMSCC-104 cells and UMSCC-47 cells with CD44shRNA or c-Jun shRNA or HPV16 E6 shRNA) appears to be more effective in tumor size reduction than chemotherapy alone. Thus, these newly-discovered HA/CD44-c-Jun/HPV16E6 signaling pathways may provide new drug targets for overcoming cisplatin chemoresistance in HPV16E6-positive OPSCC cells.

Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1.

We have previously demonstrated that phospholipase C (PLC)-gamma1 is required for calcium-induced human keratinocyte differentiation. In the present study, we investigated whether the activation of PLC-gamma1 by nonreceptor kinases such as src and fyn plays a role in mediating this process. Our results showed that the combination of dominant negative src and fyn blocked calcium-stimulated PLC-gamma1 activity and human keratinocyte differentiation, whereas each separately has little effect. However, unlike the activation of PLC-gamma1 by epidermal growth factor, calcium-induced activation of PLC-gamma1 was not a result of direct tyrosine phosphorylation. Therefore, we examined an alternative mechanism, in particular phosphatidylinositol 3,4,5-triphosphate (PIP3) formed as a product of phosphatidylinositol 3-kinase (PI3K) activity. PIP3 binds to and activates PLC-gamma1. The combination of dominant negative src and fyn blocked calcium-induced tyrosine phosphorylation of the regulatory subunit of PI3K, p85alpha, and the activity of the catalytic subunit of PI3K. PI3K inhibitors blocked calcium activation of PLC-gamma1 as well as the induction of keratinocyte differentiation markers involucrin and transglutaminase. These data indicate that calcium activates PLC-gamma1 via increased PIP3 formation mediated by c-src- and fyn-activated PI3K. This activation is required for calcium-induced human keratinocyte differentiation.

Hyaluronan-CD44 interaction stimulates keratinocyte differentiation, lamellar body formation/secretion, and permeability barrier homeostasis.

In this study we investigated whether hyaluronan (HA)-CD44 interaction influences epidermal structure and function. Our data show that CD44 deficiency is accompanied by reduction in HA staining in CD44 knockout (k/o) mouse skin leading to a marked thinning of epidermis versus wild-type mouse skin. A significant delay in the early barrier recovery (following acute barrier disruption) occurs in CD44 k/o versus wild-type mouse skin. To assess the basis for these alterations in CD44 k/o mouse epidermis, we determined that differentiation markers are greatly reduced in the epidermis of CD44 k/o versus wild-type mice, while conversely HA binding to CD44 triggers differentiation in cultured human keratinocytes. CD44 downregulation (using CD44 small interfering RNAs) also inhibits HA-mediated keratinocyte differentiation. Slower barrier recovery in CD44 k/o mice could be further attributed to reduced lamellar body formation, loss of apical polarization of LB secretion, and downregulation of cholesterol synthesis. Accordingly, HA-CD44 binding stimulates both LB formation and secretion. Together, these observations demonstrate new roles for HA-CD44 interaction in regulating both epidermal differentiation and lipid synthesis/secretion, which in turn influence permeability barrier homeostasis. HA-CD44 signaling could comprise a novel approach to treat skin disorders characterized by abnormalities in differentiation, lipid synthesis, and/or barrier function.

Hyaluronan-CD44 promotes phospholipase C-mediated Ca2+ signaling and cisplatin resistance in head and neck cancer.

OBJECTIVE: To investigate whether hyaluronan (HA)-CD44 promotes head and neck squamous cell carcinoma (HNSCC) cisplatin resistance and whether HA-CD44 promotes phospholipase C (PLC)-mediated Ca2+ signaling to alter cisplatin sensitivity in HNSCC. DESIGN: Cell line study. MAIN OUTCOME MEASURES: Tumor cell growth with the chemotherapeutic drug cisplatin was measured in the presence or absence of HA, anti-CD44 antibody plus HA, and other inhibitors of the PLC-mediated Ca2+ signaling pathway. Ca2+ mobilization was measured with fluorescence spectrophotometry using the Ca2+ binding dye Fura/2AM. RESULTS: In the absence of HA, cisplatin inhibited tumor cell growth. The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 5-fold reduced ability of cisplatin to cause HNSCC cell death, suggesting that HA can promote CD44-dependent cisplatin resistance. Fluorescence spectrophotometry demonstrated that HA can promote CD44-dependent Ca2+ mobilization in HNSCC. On the other hand, the presence of U73122, a PLC inhibitor, and 2-aminoethoxydiphenyl borate, an inositol-1,4,5-triphosphate receptor inhibitor, eliminated HA-mediated Ca2+ mobilization and HA-mediated cisplatin resistance in these cell lines. CONCLUSIONS: Our results indicate that HA-CD44 signaling influences cisplatin sensitivity in HNSCC cell growth. In particular, HA-CD44 promotion of PLC-mediated Ca2+ signaling plays a role in cisplatin resistance in HNSCC cells. Perturbation of this HA-CD44-mediated signaling pathway may be a promising target to overcome cisplatin resistance in HNSCC.

Hyaluronan and the interaction between CD44 and epidermal growth factor receptor in oncogenic signaling and chemotherapy resistance in head and neck cancer.

OBJECTIVES: To investigate whether hyaluronan (HA) and CD44 (hereinafter HA-CD44) promotes head and neck squamous cell carcinoma (HNSCC) chemotherapy resistance and whether HA-CD44 promotes epidermal growth factor receptor (EGFR)-mediated oncogenic signaling to alter chemotherapy sensitivity in HNSCC. Hyaluronan, a glycosaminoglycan component of the extracellular matrix, is a ligand for the transmembrane receptor CD44, which acts through multiple signaling pathways to influence cellular behavior. We recently determined that HA-CD44 promotes phospholipase C-mediated calcium signaling and cisplatin resistance in HNSCC. DESIGN: Cell line study. MAIN OUTCOME MEASURES: Tumor cell growth with various chemotherapeutic drugs (methotrexate, doxorubicin hydrochloride, adriamycin, and cisplatin) was measured in the presence or absence of HA and other inhibitors of the EGFR-mediated signaling pathway. Immunoblotting was used to study EGFR signaling. Migration assays provided one measure of tumor progression. RESULTS: The addition of HA, but not HA plus anti-CD44 antibody, resulted in a 2-fold reduced ability of methotrexate and an 8-fold reduced ability of adriamycin to cause HNSCC cell death. Immunoblotting studies demonstrated that HA can promote an association between CD44 and EGFR as well as CD44-dependent activation of EGFR-mediated signaling. Migration assays demonstrated that HA-CD44 can promote tumor migration with EGFR signaling. The presence of AG1478, an EGFR inhibitor, and U0126, an extracellular signal-regulated kinase inhibitor, inhibited HA-mediated tumor growth, migration, and chemotherapy resistance. CONCLUSIONS: Our results indicate that HA promotes CD44/EGFR interaction, EGFR-mediated oncogenic signaling, and chemotherapy resistance in HNSCC. Perturbation of HA-CD44-mediated signaling may be a promising and novel strategy to treat HNSCC.