RESUMO
Patients with squamous cell carcinoma (SCC) have significantly lower survival upon the development of distant metastases. The extracellular matrix (ECM) is a consistent yet dynamic influence on the metastatic capacity of SCCs. The ECM encompasses a milieu of structural proteins, signaling molecules, and enzymes. Just over 40 years ago, the fibrous ECM glycoprotein laminin was identified. Roughly four decades of research have revealed a pivotal role of laminins in metastasis. However, trends in ECM alterations in some cancers have been applied broadly to all metastatic diseases, despite evidence that these characteristics vary by tumor type. We will summarize how laminins influence the SCC metastatic process exclusively. Enhanced laminin protein deposition occurs at the invasive edge of SCC tumors, which correlates with elevated levels of laminin-binding ß1 integrins on SCC cells, increased MMP-3 presence, worse prognosis, and lymphatic dissemination. Although these findings are significant, gaps in knowledge of the formation of a premetastatic niche, the processes of intra- and extravasation, and the contributions of the ECM to SCC metastatic cell dormancy persist. Bridging these gaps requires novel in vitro systems and animal models that reproduce tumor-stromal interactions and spontaneous metastasis seen in the clinic. These advances will allow accurate assessment of laminins to predict responders to transforming growth factor-ß inhibitors and immunotherapy, as well as potential combinatorial therapies with the standard of care. Such clinical interventions may drastically improve quality of life and patient survival by explicitly targeting SCC metastasis.
Assuntos
Carcinoma de Células Escamosas , Laminina , Animais , Laminina/metabolismo , Qualidade de Vida , Carcinoma de Células Escamosas/metabolismo , Matriz Extracelular/metabolismo , Adesão CelularRESUMO
Head and neck squamous cell carcinoma (HNSCC) is commonly associated with tobacco and alcohol consumption that induce a "precancerous field," with phosphoinositide 3-kinase (PI3K) signaling being a common driver. However, the preclinical effectiveness of PI3K inhibitors has not necessarily translated to remarkable benefit in HNSCC patients. Thus, we sought to determine how precancerous keratinocytes influence HNSCC proliferation, cancer stem cell (CSC) maintenance, and response to PI3K inhibitors. We used the NOK keratinocyte cell line as a model of preneoplastic keratinocytes because it harbors two frequent genetic events in HNSCC, CDKN2A promoter methylation and TP53 mutation, but does not form tumors. NOK cell coculture or NOK cell-conditioned media promoted HNSCC proliferation, PI3K inhibitor resistance, and CSC phenotypes. SOMAscan-targeted proteomics determined the relative levels of >1300 analytes in the media conditioned by NOK cells and HNSCC cells ± PI3K inhibitor. These results demonstrated that NOK cells release abundant levels of ligands that activate epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR), two receptor tyrosine kinases with oncogenic activity. Inhibition of EGFR, but not FGFR, blunted PI3K inhibitor resistance and CSC phenotypes induced by NOK cells. Our results demonstrate that precancerous keratinocytes can directly support neighboring HNSCC by activating EGFR. Importantly, PI3K inhibitor sensitivity was not necessarily a cancer cell-intrinsic property, and the tumor microenvironment impacts therapeutic response and supports CSCs. Additionally, combined inhibition of EGFR with PI3K inhibitor diminished EGFR activation induced by PI3K inhibitor and potently inhibited cancer cell proliferation and CSC maintenance.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Lesões Pré-Cancerosas , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Queratinócitos/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Receptores de Fatores de Crescimento de Fibroblastos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Microambiente TumoralRESUMO
Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.
Assuntos
Carcinoma de Células Escamosas/etiologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Genes p53 , Neoplasias de Cabeça e Pescoço/etiologia , Queratina-15/metabolismo , Animais , Carcinoma de Células Escamosas/mortalidade , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias de Cabeça e Pescoço/mortalidade , Humanos , Linfócitos do Interstício Tumoral , Camundongos Transgênicos , Neoplasias Experimentais , Microambiente TumoralRESUMO
SMAD4 is a potent tumor suppressor and a central mediator of the TGFß signaling pathway. SMAD4 genetic loss is frequent in squamous cell carcinomas (SCCs). Reports of SMAD4 expression in SCCs vary significantly possibly due to inter-tumor heterogeneity or technical reasons. SMAD4 loss is an initiation event for SCCs. In tumor epithelial cells, SMAD4 loss causes increased proliferation, decreased apoptosis, and "Brca-like" genomic instability associated with DNA repair defects. SMAD4 loss also plays a role in the expansion of cancer stem cells. Epithelial SMAD4 loss causes overexpression of TGFß that is released into the tumor microenvironment and contributes to SCC progression through proinflammatory and immune evasive mechanisms. SMAD4 loss, while not a direct therapeutic target, is associated with multiple targetable pathways that require further therapeutic studies. Altogether, SMAD4 loss is a potential biomarker in SCCs that should be further studied for its values in prognostic and therapeutic predictions. Such information will potentially guide future biomarker-driven clinical trial designs and improve SCC patient outcomes.
Assuntos
Carcinoma de Células Escamosas/genética , Proteína Smad4/genética , Animais , Biomarcadores Tumorais/genética , Reparo do DNA/genética , Células Epiteliais/patologia , Genes Supressores de Tumor/fisiologia , Humanos , Transdução de Sinais/genética , Microambiente Tumoral/genéticaRESUMO
Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism.
Assuntos
Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Animais/patologia , Morfogênese/genética , Neuropeptídeos/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Feminino , Neoplasias Mamárias Animais/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese/fisiologia , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Técnicas de Cultura de Órgãos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear. METHODS: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays. RESULTS: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility. CONCLUSION: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Amplificação de Genes , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genética , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismoRESUMO
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
Assuntos
Neoplasias da Mama/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mutação/genética , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais , Anfirregulina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Classe I de Fosfatidilinositol 3-Quinases , Intervalo Livre de Doença , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/antagonistas & inibidores , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Feminino , Humanos , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteômica , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas em Tandem , Regulação para Cima/efeitos dos fármacosRESUMO
Human epidermal growth factor receptor 2 (HER2; ERBB2) amplification and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations often co-occur in breast cancer. Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway has been shown to correlate with a diminished response to HER2-directed therapies. We generated a mouse model of HER2-overexpressing (HER2(+)), PIK3CA(H1047R)-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared with mice expressing either oncogene alone. HER2 and mutant PIK3CA also cooperated to promote lung metastases. By microarray analysis, HER2-driven tumors clustered with luminal breast cancers, whereas mutant PIK3CA tumors were associated with claudin-low breast cancers. PIK3CA and HER2(+)/PIK3CA tumors expressed elevated transcripts encoding markers of epithelial-to-mesenchymal transition and stem cells. Cells from HER2(+)/PIK3CA tumors more efficiently formed mammospheres and lung metastases. Finally, HER2(+)/PIK3CA tumors were resistant to trastuzumab alone and in combination with lapatinib or pertuzumab. Both drug resistance and enhanced mammosphere formation were reversed by treatment with a PI3K inhibitor. In sum, PIK3CA(H1047R) accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers, and generates resistance to approved combinations of anti-HER2 therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Genes erbB-2 , Neoplasias Mamárias Experimentais/patologia , Mutação , Fosfatidilinositol 3-Quinases/genética , Animais , Classe I de Fosfatidilinositol 3-Quinases , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos TransgênicosRESUMO
INTRODUCTION: Human epidermal growth factor receptor-2 (HER2) gene amplification (HER2+) drives tumor cell growth and survival in ~25% of breast cancers. HER2 signaling activates the type I phosphoinositide 3-kinase (PI3K), upon which these tumors rely. Consequently, inhibitors of HER2 and type I PI3K block growth and increase apoptosis in HER2+ breast cancers, especially when used in combination. However, the impact of type III PI3K inhibition, particularly in combination with HER2 blockade or type I PI3K inhibition, remains less clear. METHODS: We utilized small molecule kinase inhibitors, locked nucleic acid antisense oligonucleotides (LNA-ASOs), and siRNA to assess proliferation, autophagy, apoptosis, and protein expression in cell culture models of HER2+ breast cancers. RESULTS: Treatment of HER2+ breast cancer cells with HER2 inhibitors or type I PI3K kinase inhibitors, alone or in combination, blocked type I PI3K signaling, reduced tumor cell growth, and induced autophagy. Knockdown of the type I PI3K, p110α, using an LNA-ASO termed EZN4150 inhibited PI3K-mediated Akt phosphorylation. However, in contrast to catalytic inhibitors of type I PI3Ks, EZN4150 did not induce autophagy, and blocked autophagy in response to inhibitors of HER2 or type I PI3Ks in a dominant fashion. Sequence analysis of EZN4150 revealed significant homology to the gene encoding the type III PI3K, Vps34, a key component for autophagy induction. EZN4150 simultaneously reduced expression of both p110α and Vps34. Combined inhibition of PI3K signaling and autophagy using individual siRNAs against p110α and Vps34 or using pharmacological type I and type III PI3K inhibitors recapitulated what was seen with EZN4150, and robustly enhanced tumor cell killing. CONCLUSIONS: These studies highlight the important role of Vps34-mediated autophagy in limiting the anti-tumor response to inhibitors of HER2 or type I PI3K in HER2+ breast cancers. The type III PI3K Vps34 represents a potential therapeutic target to block treatment-induced autophagy and enhance tumor cell killing.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/metabolismo , Autofagia/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Oral squamous cell carcinoma (OSCC) is a devastating disease most often associated with tobacco consumption that induces a field of mutations from which a tumor arises. Identification of ways to prevent the emergence of cancer in high-risk patients is an ultimate goal for combatting all types of cancer, including OSCC. METHODS: Our study employs a mouse model of tongue carcinogenesis induced by tobacco carcinogen mimetic, 4-nitroquinoline 1-oxide (4NQO), to establish tongue dysplasia and OSCC. We use conventional histology, immunohistochemistry, multispectral imaging, mass cytometry, novel cell lines, pharmaceutical inhibition of PI3Kγ, T-cell suppression assays and mouse transplant models in our functional experimentation. RESULTS: In our study, we identify Ly6G+ granulocytes as the most abundant immune cell type in a model of tongue carcinogenesis induced by tobacco carcinogen mimetic 4NQO. Targeting Ly6G+ granulocytes with a pharmacologic inhibitor of PI3Kγ, an isoform of PI3K exclusively expressed by myeloid cells, resulted in reduced tongue dysplasia severity, and reduced rates of OSCC. Importantly, we performed functional assays with the Ly6G+ granulocytes induced in cell line models of 4NQO carcinogenesis to demonstrate that these granulocytes have increased polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) activity against T-cell proliferation and these PMN-MDSCs play a functional role in promoting tumor formation by inhibiting tumor regression in a PI3Kγ-dependent manner. CONCLUSIONS: Overall, our data suggest that recruitment of PMN-MDSCs to sites of dysplasia is critical to immune suppression of CD8 T cells, thereby permitting malignancy, and PI3Kγ inhibitors are one mechanism to reduce PMN-MDSC recruitment, immunosuppression and tumorigenesis in OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Células Supressoras Mieloides , Humanos , Animais , Camundongos , Fosfatidilinositol 3-Quinase , Carcinoma de Células Escamosas/induzido quimicamente , Neoplasias Bucais/induzido quimicamente , Carcinogênese , Carcinógenos/toxicidade , Carcinoma de Células Escamosas de Cabeça e Pescoço , FosfatidilinositóisRESUMO
Long-term management of inflammatory skin diseases is challenging because of side effects from repeated use of systemic treatments or topical corticosteroids. This study sought to identify the mechanisms and developmental therapeutics for these diseases using genetic models and pharmacological approaches. We found that mice overexpressing SMAD7 in keratinocytes but not mice overexpressing the N-terminal domain of SMAD7 (i.e., N-SMAD7) were resistant to imiquimod-induced T helper 1/17- and T helper 2-type inflammation. We generated a Tat-PYC-SMAD7 (truncated SMAD7 protein encompassing C-terminal SMAD7 and PY motif fused with cell-penetrating Tat peptide). Topically applied Tat-PYC-SMAD7 to inflamed skin entered cells upon contact and attenuated imiquimod-, 2,4-dinitrofluorobenzene-, and tape-stripping-induced inflammation. RNA-sequencing analyses of mouse skin exposed to these insults showed that in addition to inhibiting TGFß/NF-κB, SMAD7 blunted IL-22/signal transducer and activator of transcription 3 activation and associated pathogenesis, which is due to SMAD7 transcriptionally upregulating IL-22 antagonist IL-22RA2. Mechanistically, SMAD7 facilitated nuclear translocation and DNA binding of C/EBPß to IL22RA2 promoter for IL22RA2 transactivation. Consistent with the observations in mice mentioned earlier, transcript levels of IL22RA2 were increased in human atopic dermatitis and psoriasis lesions with clinical remission. Our study identified the anti-inflammation functional domain of SMAD7 and suggests the mechanism and feasibility for developing SMAD7-based biologics as a topical therapy for skin inflammatory disorders.
Assuntos
Dermatite , Psoríase , Receptores de Interleucina , Camundongos , Humanos , Animais , Imiquimode/farmacologia , Proteína Smad7/genética , Pele/patologia , Psoríase/tratamento farmacológico , Psoríase/genética , Psoríase/induzido quimicamente , Dermatite/patologia , Queratinócitos/metabolismo , Inflamação/patologia , Fenótipo , Modelos Animais de Doenças , Camundongos Endogâmicos BALB CRESUMO
Transforming growth factor beta (TGFß) activity is linked to metastasis in many cancer types, but whether TGFß activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4-/- SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFß receptor (TGFßR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFß activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Humanos , Camundongos , Animais , Óxido Nítrico Sintase Tipo II , Fator de Crescimento Transformador beta/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/metabolismo , Células Mieloides/metabolismo , Óxido Nítrico/metabolismoRESUMO
PURPOSE: Recent studies reported therapeutic effects of Smad7 on oral mucositis in mice without compromising radiation therapy-induced cancer cell killing in neighboring oral cancer. This study aims to assess whether a Smad7-based biologic can treat oral mucositis in a clinically relevant setting by establishing an oral mucositis model in dogs and analyzing molecular targets. METHODS AND MATERIALS: We created a truncated human Smad7 protein fused with the cell-penetrating Tat tag (Tat-PYC-Smad7). We used intensity modulated radiation therapy to induce oral mucositis in dogs and applied Tat-PYC-Smad7 to the oral mucosa in dose-finding studies after intensity modulated radiation therapy. Clinical outcomes were evaluated. Molecular targets were analyzed in biopsies and serum samples. RESULTS: Tat-PYC-Smad7 treatment significantly shortened the duration of grade 3 oral mucositis based on double-blinded Veterinary Radiation Therapy Oncology Group scores and histopathology evaluations. Topically applied Tat-PYC-Smad7 primarily penetrated epithelial cells and was undetectable in serum. NanoString nCounter Canine IO Panel identified that, compared to the vehicle samples, top molecular changes in Tat-PYC-Smad7 treated samples include reductions in inflammation and cell death and increases in cell growth and DNA repair. Consistently, immunostaining shows that Tat-PYC-Smad7 reduced DNA damage and neutrophil infiltration with attenuated TGF-ß and NFκB signaling. Furthermore, IL-1ß and TNF-α were lower in Tat-PYC-Smad7 treated mucosa and serum samples compared to those in vehicle controls. CONCLUSIONS: Topical Tat-PYC-Smad7 application demonstrated therapeutic effects on oral mucositis induced by intensity modulated radiation therapy in dogs. The local effects of Tat-PYC-Smad7 targeted molecules involved in oral mucositis pathogenesis as well as reduced systemic inflammatory cytokines.
Assuntos
Mucosite , Lesões por Radiação , Estomatite , Animais , Cães , Produtos do Gene tat/metabolismo , Camundongos , Lesões por Radiação/complicações , Proteína Smad7/genética , Proteína Smad7/metabolismo , Estomatite/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Transforming growth factor beta (TGFß) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFß and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFß inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFß and PD-L1 to treat mouse SCCs and found TGFß inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFß and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.
Assuntos
Antígeno B7-H1/genética , Carcinoma de Células Escamosas/imunologia , Fator de Crescimento Transformador beta/genética , Microambiente Tumoral/imunologia , Animais , Carcinoma de Células Escamosas/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta/metabolismoRESUMO
Cancer-associated fibroblasts (CAFs) have been shown to enhance squamous cell carcinoma (SCC) growth, but it is unclear whether they promote SCC lung metastasis. We generated CAFs from K15.KrasG12D.Smad4-/- mouse SCCs. RNA expression analyses demonstrated that CAFs had enriched transforming growth factor-beta (TGFß) signaling compared to normal tissue-associated fibroblasts (NAFs), therefore we assessed how TGFß-enriched CAFs impact SCC metastasis. We co-injected SCC cells with CAFs to the skin, tail vein, or the lung to mimic sequential steps of lung metastasis. CAFs increased SCC volume only in lung co-transplantations, characterized with increased proliferation and angiogenesis and decreased apoptosis compared to NAF co-transplanted SCCs. These CAF effects were attenuated by a clinically relevant TGFß receptor inhibitor, suggesting that CAFs facilitated TGFß-dependent SCC cell seeding and survival in the lung. CAFs also increased tumor volume when co-transplanted to the lung with limiting numbers of SCC cancer stem cells (CSCs). In vitro, CSC sphere formation and invasion were increased either with co-cultured CAFs or with CAF conditioned media (which contains the highest TGFß1 concentration) and these CAF effects were blocked by TGFß inhibition. Further, TGFß activation was higher in primary human oral SCCs with lung metastasis than SCCs without lung metastasis. Similarly, TGFß activation was detected in the lungs of mice with micrometastasis. Our data suggest that TGFß-enriched CAFs play a causal role in CSC seeding and expansion in the lung during SCC metastasis, providing a prognostic marker and therapeutic target for SCC lung metastasis.
RESUMO
Transforming growth factor-ß (TGF-ß) signaling is essential in embryo development and maintaining normal homeostasis. Extensive evidence shows that TGF-ß activation acts on several cell types, including epithelial cells, fibroblasts, and immune cells, to form a pro-fibrotic environment, ultimately leading to fibrotic diseases. TGF-ß is stored in the matrix in a latent form; once activated, it promotes a fibroblast to myofibroblast transition and regulates extracellular matrix (ECM) formation and remodeling in fibrosis. TGF-ß signaling can also promote cancer progression through its effects on the tumor microenvironment. In cancer, TGF-ß contributes to the generation of cancer-associated fibroblasts (CAFs) that have different molecular and cellular properties from activated or fibrotic fibroblasts. CAFs promote tumor progression and chronic tumor fibrosis via TGF-ß signaling. Fibrosis and CAF-mediated cancer progression share several common traits and are closely related. In this review, we consider how TGF-ß promotes fibrosis and CAF-mediated cancer progression. We also discuss recent evidence suggesting TGF-ß inhibition as a defense against fibrotic disorders or CAF-mediated cancer progression to highlight the potential implications of TGF-ß-targeted therapies for fibrosis and cancer.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Progressão da Doença , Fibrose , Humanos , Terapia de Alvo MolecularRESUMO
Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.
Assuntos
Neoplasias de Cabeça e Pescoço/patologia , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Macrófagos Associados a Tumor/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Retroalimentação Fisiológica , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Ácido Hialurônico/metabolismo , Masculino , Camundongos Endogâmicos NOD , Monócitos/metabolismo , Monócitos/patologia , Células-Tronco Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Microambiente Tumoral , Macrófagos Associados a Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
PURPOSE: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). EXPERIMENTAL DESIGN: We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. RESULTS: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. CONCLUSIONS: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Modelos Animais de Doenças , Neoplasias de Cabeça e Pescoço/radioterapia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteína Smad4/deficiência , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Animais , Apoptose , Proliferação de Células , Cetuximab/administração & dosagem , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Nus , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The high rate of glucose utilization by cancer cells has been well characterized. Recent data suggest that when normal mammary epithelial cells are cultured under nonadherent conditions, glucose consumption decreases, ATP levels fall, and concentrations of reactive oxygen species rise. The rise in reactive oxygen species causes death of nonadherent cells, which can be suppressed with antioxidants. Nonadherent ErbB2-transformed mammary epithelial cells maintain glucose transport and antioxidant production; however, antioxidants appear to enhance anchorage-independent growth. These findings integrate aspects of glucose metabolism, anoikis suppression and antioxidant production in tumor cell biology and suggest that antioxidant therapy could stimulate tumor survival.
Assuntos
Neoplasias da Mama/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias da Mama/patologia , Adesão Celular/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
PURPOSE: We previously reported preventive and therapeutic effects of Smad7, a multifunctional protein, on radiotherapy (RT)-induced mucositis in mice without promoting human oral cancer cell survival or migration in vitro. The current study aims to determine whether a Smad7-based biologic can treat existing oral mucositis during radiotherapy for oral cancer and whether this treatment compromises RT-induced cancer cell killing in neighboring oral cancer.Experimental Design: We transplanted human oral cancer cells into the tongues of mice and applied craniofacial irradiation to simultaneously kill tumor cells and induce oral mucositis, thus modeling RT and mucositis in oral cancer patients. We topically applied a recombinant human Smad7 protein fused with the cell-penetrating Tat tag (Tat-Smad7) to the oral mucosa of tumor-bearing mice post RT when oral mucositis began to develop. RESULTS: Topically applied Tat-Smad7 penetrated cells in both the oral mucosa and oral cancer, attenuating TGFß and NF-κB signaling as well as inflammation at both sites. Tat-Smad7 treatment alleviated oral mucositis with reductions in DNA damage and apoptosis in keratinocytes, but increased keratinocyte proliferation compared with vehicle-treated mucositis lesions. In contrast, adjacent oral cancer exposed to Tat-Smad7 did not show alterations in proliferation or direct DNA damage, but showed increased oxidative stress damage and apoptosis compared with tumors treated with vehicle. CONCLUSIONS: Our results suggest that short-course Tat-Smad7 application to oral mucositis promotes its healing but does not compromise the cytotoxic effect of RT on oral cancer and has context-specific effects on oral mucosa versus oral cancer.