ABSTRACT
The solid tumour microenvironment includes nerve fibres that arise from the peripheral nervous system1,2. Recent work indicates that newly formed adrenergic nerve fibres promote tumour growth, but the origin of these nerves and the mechanism of their inception are unknown1,3. Here, by comparing the transcriptomes of cancer-associated trigeminal sensory neurons with those of endogenous neurons in mouse models of oral cancer, we identified an adrenergic differentiation signature. We show that loss of TP53 leads to adrenergic transdifferentiation of tumour-associated sensory nerves through loss of the microRNA miR-34a. Tumour growth was inhibited by sensory denervation or pharmacological blockade of adrenergic receptors, but not by chemical sympathectomy of pre-existing adrenergic nerves. A retrospective analysis of samples from oral cancer revealed that p53 status was associated with nerve density, which was in turn associated with poor clinical outcomes. This crosstalk between cancer cells and neurons represents mechanism by which tumour-associated neurons are reprogrammed towards an adrenergic phenotype that can stimulate tumour progression, and is a potential target for anticancer therapy.
Subject(s)
Adrenergic Neurons/pathology , Cell Transdifferentiation , Cellular Reprogramming , Mouth Neoplasms/pathology , Sensory Receptor Cells/pathology , Tumor Suppressor Protein p53/deficiency , Adrenergic Antagonists/pharmacology , Adrenergic Antagonists/therapeutic use , Animals , Cell Division , Disease Models, Animal , Disease Progression , Female , Humans , Male , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , Mouth Neoplasms/drug therapy , Mouth Neoplasms/genetics , Mouth Neoplasms/metabolism , Nerve Fibers/pathology , Neurites/pathology , Receptors, Adrenergic/metabolism , Retrospective Studies , Tumor Microenvironment , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor AssaysABSTRACT
Alterations of the tumor suppressor TP53, one of the most common events in cancer, alone are insufficient for tumor development but serve as drivers of transformation. We sought to identify cooperating events through genomic analyses of a somatic Trp53R245W mouse model (equivalent to the TP53R248W hot spot mutation in human cancers) that recapitulates metastatic breast-cancer development. We identified cooperating lesions similar to those found in human breast cancers. Moreover, we identified activation of the Pi3k/Akt/mTOR pathway in most tumors via mutations in Pten, Erbb2, Kras, and/or a recurrent Pip5k1c mutation that stabilizes the Pip5k1c protein and activates Pi3k/Akt/mTOR signaling. Another PIP5K1C family member, PIP5K1A, is coamplified with PI4KB in 18% of human breast cancer patients; both encode kinases that are responsible for production of the PI3K substrate, phosphatidylinositol 4,5-bisphosphate. Thus, the TP53R248W mutation and PI3K/AKT/mTOR signaling are major cooperative events driving breast-cancer development. Additionally, a combination of two US Food and Drug Administration (FDA)-approved drugs, tigecycline and metformin, which target oxidative phosphorylation downstream of PI3K signaling, inhibited tumor cell growth and may be repurposed for breast-cancer treatment. These findings advance our understanding of how mutant p53 drives breast-tumor development and pinpoint the importance of PI3K/AKT/mTOR signaling, expanding combination therapies for breast-cancer treatment.
Subject(s)
Breast Neoplasms , Phosphatidylinositol 3-Kinases , Animals , Female , Humans , Mice , Breast Neoplasms/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: The treatment of advanced oral squamous cell carcinoma (OSCC) is a clinical challenge because it is unclear which therapeutic approaches are the best for this highly heterogeneous group of patients. Because TP53 mutations are the most common genetic event in these tumors, the authors investigated whether they could represent an ancillary biomarker in the management of advanced OSCC. METHODS: The TP53 gene was sequenced in 78 samples from patients with advanced OSCC who received treatment at 2 institutions located in the United States and Brazil. TP53 mutations were classified according to an in-silico impact score (the evolutionary action score of p53 [EAp53]), which identifies mutations that have greater alterations of p53 protein function (high-risk). Associations between TP53 mutation status/characteristics and clinicopathologic characteristics were investigated. The relevant findings were validated in silico by analyzing 197 samples from patients with advanced OSCC from The Cancer Genome Atlas. RESULTS: No differences in clinical outcomes were detected between patients with TP53-mutant and wild-type TP53 disease. However, patients who had tumors carrying high-risk TP53 mutations had a significantly increased risk of developing extranodal extension (ENE) compared with those who had wild-type TP53-bearing tumors. The increased chances of detecting ENE among patients who had high-risk TP53 mutations was validated among patients with advanced OSCC from The Cancer Genome Atlas cohort. CONCLUSIONS: High-risk TP53 mutations are associated with an increased chance of detecting ENE in patients with advanced OSCC. Because ENE is 1 of the major factors considered for OSCC patient management, TP53 mutation status may represent a potential ancillary biomarker for treatment decisions regarding postoperative adjuvant therapy.
Subject(s)
Mouth Neoplasms/genetics , Mutation , Squamous Cell Carcinoma of Head and Neck/genetics , Tumor Suppressor Protein p53/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Female , Humans , Male , Middle Aged , Mouth Neoplasms/metabolism , Mouth Neoplasms/pathology , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Extranodal extension (ENE) of lymph node metastasis is one of the most reliable prognostic indicators for patients with locally advanced oral cancer. Although multiple reports have found a close relationship between immune infiltration of tumors and patient clinical outcomes, its association with ENE is unknown. METHODS: We identified 234 human papillomavirus-negative (HPV-) oral cavity squamous cell carcinoma (OSCC) patients in The Cancer Genome Atlas and investigated the immune infiltration profiles of primary tumors and their association with survival. RESULTS: Hierarchical clustering analysis clearly classified the overall immune infiltration status in OSCC into high immune or low immune groups. The combination of ENE positivity and low immune infiltration was strongly associated with poor overall survival (OS) compared to the combination of ENE positivity and high immune infiltration [hazard ratio 2.04 (95 %CI, 1.08-3.83); p = 0.024]. The immune infiltration status was not associated with OS rates in patients with ENE-negative or node negative tumors. CONCLUSION: Overall Immune infiltration at the primary site was significantly associated with clinical outcome of OSCC patients with ENE.
Subject(s)
Lymphatic Metastasis , Mouth Neoplasms , Humans , Mouth Neoplasms/pathology , Mouth Neoplasms/immunology , Mouth Neoplasms/mortality , Male , Female , Middle Aged , Aged , Carcinoma, Squamous Cell/immunology , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/mortality , Prognosis , Extranodal Extension/pathology , AdultABSTRACT
Metastatic disease remains the leading cause of death due to cancer, yet the mechanism(s) of metastasis and its timely detection remain to be elucidated. Neutrophil elastase (NE), a serine protease secreted by neutrophils, is a crucial mediator of chronic inflammation and tumor progression. In this study, we used the PyMT model (NE+/+ and NE-/-) of breast cancer to interrogate the tumor-intrinsic and -extrinsic mechanisms by which NE can promote metastasis. Our results showed that genetic ablation of NE significantly reduced lung metastasis and improved metastasis-free survival. RNA-sequencing analysis of primary tumors indicated differential regulation of tumor-intrinsic actin cytoskeleton signaling pathways by NE. These NE-regulated pathways are critical for cell-to-cell contact and motility and consistent with the delay in metastasis in NE-/- mice. To evaluate whether pharmacologic inhibition of NE inhibited pulmonary metastasis and phenotypically mimicked PyMT NE-/- mice, we utilized AZD9668, a clinically available and specific NE inhibitor. We found AZD9668 treated PyMT-NE+/+ mice showed significantly reduced lung metastases, improved recurrence-free, metastasis-free and overall survival, and their tumors showed similar molecular alterations as those observed in PyMT-NE-/- tumors. Finally, we identified a NE-specific signature that predicts recurrence and metastasis in patients with breast cancer. Collectively, our studies suggest that genetic ablation and pharmacologic inhibition of NE reduces metastasis and extends survival of mouse models of breast cancer, providing rationale to examine NE inhibitors as a treatment strategy for the clinical management of patients with metastatic breast cancer.
Subject(s)
Breast Neoplasms , Lung Neoplasms , Pyridones , Sulfones , Animals , Female , Humans , Mice , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Leukocyte Elastase/genetics , Lung Neoplasms/pathologyABSTRACT
The incidence of human papilloma virus-mediated oropharyngeal squamous cell carcinoma (OPSCC) has increased over the past 40 years, particularly among young individuals with a favorable prognosis; however, current therapy often leads to unfortunate side effects, such as dysphagia. Despite the emphasis on dysphagia in previous studies, there is an important research gap in understanding the correlation between neuronal changes and patient-reported and functional outcomes in patients with OPSCC. To address this issue, we examined pathologic tissue samples from patients with OPSCC using multiplex immunofluorescence staining and machine learning to correlate tumor-associated neuronal changes with prospectively collected patient-reported and functional outcomes. We found that tumor enrichment of adrenergic (TH+) and CGRP+ sensory-afferent nerves correlated with poorer swallowing outcomes. Functional electromyography recordings showed correlations between growing (GAP43+) and immature cholinergic (ChAT+DCX+) nerves and denervation patterns in survivors of OPSCC. A murine model of radiation-induced dysphagia further confirmed that immature cholinergic and CGRP+ nerves were correlated with impaired swallowing. Preclinical interventional studies also supported the independent contributions of CGRP+ and cholinergic (ChAT+) nerves to dysphagia in treated mouse models of OPSCC. Our results suggest that CGRP+ and ChAT+ neuronal signaling play distinct roles in tumor- and radiation-induced dysphagia in OPSCC and offer a comprehensive dataset on the neural landscape of OPSCC. These insights may guide early interventions for swallow preservation and the repurposing of neurology-related drugs, such as CGRP blockers, in clinical oncology and survivorship.
Subject(s)
Carcinoma, Squamous Cell , Deglutition Disorders , Oropharyngeal Neoplasms , Humans , Oropharyngeal Neoplasms/radiotherapy , Oropharyngeal Neoplasms/pathology , Animals , Carcinoma, Squamous Cell/radiotherapy , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/physiopathology , Male , Mice , Deglutition/radiation effects , Female , Middle Aged , Treatment Outcome , Calcitonin Gene-Related Peptide/metabolismABSTRACT
Cyclin-dependent kinases 4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) is standard of care for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC). However, resistance to CDK4/6is plus ET remains a clinical problem with limited therapeutic options following disease progression. Different CDK4/6is might have distinct mechanisms of resistance, and therefore using them sequentially or targeting their differentially altered pathways could delay disease progression. To understand pathways leading to resistance to the CDK4/6is palbociclib and abemaciclib, we generated multiple in vitro models of palbociclib-resistant (PR) and abemaciclib-resistant (AR) cell lines as well as in vivo patient-derived xenografts (PDX) and ex vivo PDX-derived organoids (PDxO) from patients who progressed on CDK4/6i. PR and AR breast cancer cells exhibited distinct transcriptomic and proteomic profiles that sensitized them to different classes of inhibitors; PR cells upregulated G2-M pathways and responded to abemaciclib, while AR cells upregulated mediators of the oxidative phosphorylation pathway (OXPHOS) and responded to OXPHOS inhibitors. PDX and organoid models derived from patients with PR breast cancer remained responsive to abemaciclib. Resistance to palbociclib while maintaining sensitivity to abemaciclib was associated with pathway-specific transcriptional activity but was not associated with any individual genetic alterations. Finally, data from a cohort of 52 patients indicated that patients with HR-positive/HER2-negative MBC who progressed on palbociclib-containing regimens can exhibit a meaningful overall clinical benefit from abemaciclib-based therapy when administered after palbociclib. These findings provide the rationale for clinical trials evaluating the benefit of abemaciclib treatment following progression on a prior CDK4/6i. SIGNIFICANCE: Palbociclib-resistant breast cancers respond to abemaciclib and express pathway-specific signatures of sensitivity, providing a biomarker-driven therapeutic option for patients with metastatic breast cancer following disease progression on cyclin-dependent kinases 4/6 inhibitors.
Subject(s)
Breast Neoplasms , Animals , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Proteomics , Disease Models, Animal , Disease Progression , Cyclins , Cyclin-Dependent Kinase 4 , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Cyclin-Dependent Kinase 6 , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic useABSTRACT
While the nervous system has reciprocal interactions with both cancer and the immune system, little is known about the potential role of tumor associated nerves (TANs) in modulating anti-tumoral immunity. Moreover, while peri-neural invasion is a well establish poor prognostic factor across cancer types, the mechanisms driving this clinical effect remain unknown. Here, we provide clinical and mechniastic association between TANs damage and resistance to anti-PD-1 therapy. Using electron microscopy, electrical conduction studies, and tumor samples of cutaneous squamous cell carcinoma (cSCC) patients, we showed that cancer cells can destroy myelin sheath and induce TANs degeneration. Multi-omics and spatial analyses of tumor samples from cSCC patients who underwent neoadjuvant anti-PD-1 therapy demonstrated that anti-PD-1 non-responders had higher rates of peri-neural invasion, TANs damage and degeneration compared to responders, both at baseline and following neoadjuvant treatment. Tumors from non-responders were also characterized by a sustained signaling of interferon type I (IFN-I) - known to both propagate nerve degeneration and to dampen anti-tumoral immunity. Peri-neural niches of non-responders were characterized by higher immune activity compared to responders, including immune-suppressive activity of M2 macrophages, and T regulatory cells. This tumor promoting inflammation expanded to the rest of the tumor microenvironment in non-responders. Anti-PD-1 efficacy was dampened by inducing nerve damage prior to treatment administration in a murine model. In contrast, anti-PD-1 efficacy was enhanced by denervation and by interleukin-6 blockade. These findings suggested a potential novel anti-PD-1 resistance drived by TANs damage and inflammation. This resistance mechanism is targetable and may have therapeutic implications in other neurotropic cancers with poor response to anti-PD-1 therapy such as pancreatic, prostate, and breast cancers.
ABSTRACT
Although cell-based studies have shown that γ-tocotrienol (γTE) exhibits stronger anticancer activities than other forms of vitamin E including γ-tocopherol (γT), the molecular bases underlying γTE-exerted effects remains to be elucidated. Here we showed that γTE treatment promoted apoptosis, necrosis and autophagy in human prostate PC-3 and LNCaP cancer cells. In search of potential mechanisms of γTE-provoked effects, we found that γTE treatment led to marked increase of intracellular dihydroceramide and dihydrosphingosine, the sphingolipid intermediates in de novo sphingolipid synthesis pathway but had no effects on ceramide or sphingosine. The elevation of these sphingolipids by γTE preceded or coincided with biochemical and morphological signs of cell death and was much more pronounced than that induced by γT, which accompanied with much higher cellular uptake of γTE than γT. The importance of sphingolipid accumulation in γTE-caused fatality was underscored by the observation that dihydrosphingosine and dihydroceramide potently reduced the viability of both prostate cell lines and LNCaP cells, respectively. In addition, myriosin, a specific inhibitor of de novo sphingolipid synthesis, counteracted γTE-induced cell death. In agreement with these cell-based studies, γTE inhibited LNCaP xenograft growth by 53% (p < 0.05), compared to 33% (p = 0.07) by γT, in nude mice. These findings provide a molecular basis of γTE-stimulated cancer cell death and support the notion that elevation of intracellular dihydroceramide and dihydrosphingosine is likely a novel anticancer mechanism.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Autophagy , Ceramides/metabolism , Chromans/pharmacology , Prostatic Neoplasms/metabolism , Sphingosine/analogs & derivatives , Vitamin E/analogs & derivatives , Animals , Cell Line, Tumor , Cell Survival , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Male , Mevalonic Acid/pharmacology , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Sphingosine/metabolism , Tumor Burden/drug effects , Vitamin E/pharmacologyABSTRACT
Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.
ABSTRACT
Immune checkpoint inhibitors (ICI) targeting CTLA4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events, including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which monoallelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe ECG abnormalities, closely recapitulating the clinical and pathologic hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI myocarditis. SIGNIFICANCE: We provide a preclinical model of ICI-associated myocarditis which recapitulates this clinical syndrome. Using this model, we demonstrate that CTLA4 and PD-1 (ICI targets) functionally interact for myocarditis development and that intervention with CTLA4-Ig (abatacept) attenuates myocarditis, providing mechanistic rationale and preclinical support for therapeutic clinical studies.See related commentary by Young and Bluestone, p. 537.This article is highlighted in the In This Issue feature, p. 521.
Subject(s)
Immune Checkpoint Inhibitors/adverse effects , Molecular Targeted Therapy/adverse effects , Myocarditis/diagnosis , Myocarditis/etiology , Neoplasms/complications , Animals , Biomarkers, Tumor/antagonists & inhibitors , Cardiotoxicity , Disease Management , Disease Models, Animal , Disease Susceptibility , Electrocardiography , Humans , Immune Checkpoint Inhibitors/therapeutic use , Mice , Myocarditis/metabolism , Neoplasms/drug therapy , Neoplasms/etiologyABSTRACT
C-type lectins play key roles in pathogen recognition, innate immunity, and cell-cell interactions. Here, we report a new C-type lectin (C-type lectin 1) from the shrimp Litopenaeus vannamei (LvCTL1), which has activity against the white spot syndrome virus (WSSV). LvCTL1 is a 156-residue polypeptide containing a C-type carbohydrate recognition domain with an EPN (Glu(99)-Pro(100)-Asn(101)) motif that has a predicted ligand binding specificity for mannose. Reverse transcription-PCR analysis revealed that LvCTL1 mRNA was specifically expressed in the hepatopancreas of L. vannamei. Recombinant LvCTL1 (rLvCTL1) had hemagglutinating activity and ligand binding specificity for mannose and glucose. rLvCTL1 also had a strong affinity for WSSV and interacted with several envelope proteins of WSSV. Furthermore, we showed that the binding of rLvCTL1 to WSSV could protect shrimps from viral infection and prolong the survival of shrimps against WSSV infection. Our results suggest that LvCTL1 is a mannose-binding C-type lectin that binds to envelope proteins of WSSV to exert its antiviral activity. To our knowledge, this is the first report of a shrimp C-type lectin that has direct anti-WSSV activity.
Subject(s)
Animal Diseases/prevention & control , Antiviral Agents/pharmacology , Lectins, C-Type/metabolism , Penaeidae/virology , White spot syndrome virus 1/drug effects , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Gene Expression Profiling , Glucose/metabolism , Hepatopancreas/metabolism , Mannose/metabolism , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Survival AnalysisABSTRACT
The NF-kappaB/IkappaBalpha pathway plays an important role in the regulation of immune and inflammatory responses. IkappaBalpha is an inhibitory molecule that sequesters transcription activator NF-kappaB dimer in the cytoplasm of unstimulated cells. Here, we isolated the full-length cDNAs of the mandarin fish (Siniperca chuatsi) alpha inhibitor of NF-kappaB (ScIkappaBalpha) and p65 NF-kappaB (Scp65). Multiple sequence alignments showed that the amino acid sequences of both ScIkappaBalpha and Scp65 contain conserved domains similar to those of mammalian counterparts. Protein pull-down and coimmunoprecipitation assays showed that ScIkappaBalpha directly bound with Scp65. Real-time quantitative PCR analysis showed that ScIkappaBalpha mRNA was constitutive in all mandarin fish tissues detected. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the mRNA level of ScIkappaBalpha was decreased nearly 6 fold in the spleen. This result suggests that the NF-kappaB/IkappaBalpha pathway in mandarin fish may play a role in the immune response against ISKNV.
Subject(s)
Fish Diseases/metabolism , I-kappa B Proteins/metabolism , Perciformes/genetics , Perciformes/metabolism , Transcription Factor RelA/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA Virus Infections/metabolism , Gene Expression Profiling , Gene Expression Regulation , I-kappa B Proteins/chemistry , Iridoviridae/physiology , Models, Molecular , Molecular Sequence Data , NF-KappaB Inhibitor alpha , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Transcription Factor RelA/chemistryABSTRACT
To address the need for improved systemic therapy for non-small-cell lung cancer (NSCLC), we previously demonstrated that mesenchymal NSCLC was sensitive to polo-like kinase (Plk1) inhibitors, but the mechanisms of resistance in epithelial NSCLC remain unknown. Here, we show that cMet was differentially regulated in isogenic pairs of epithelial and mesenchymal cell lines. Plk1 inhibition inhibits cMet phosphorylation only in mesenchymal cells. Constitutively active cMet abrogates Plk1 inhibitor-induced apoptosis. Likewise, cMet silencing or inhibition enhances Plk1 inhibitor-induced apoptosis. Cells with acquired resistance to Plk1 inhibitors are more epithelial than their parental cells and maintain cMet activation after Plk1 inhibition. In four animal NSCLC models, mesenchymal tumors were more sensitive to Plk1 inhibition alone than were epithelial tumors. The combination of cMet and Plk1 inhibition led to regression of tumors that did not regrow when drug treatment was stopped. Plk1 inhibition did not affect HGF levels but did decrease vimentin phosphorylation, which regulates cMet phosphorylation via ß1-integrin. This research defines a heretofore unknown mechanism of ligand-independent activation of cMet downstream of Plk1 and an effective combination therapy.
Subject(s)
Apoptosis , Cell Cycle Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-met/metabolism , Proto-Oncogene Proteins/antagonists & inhibitors , Vimentin/metabolism , Animals , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition/drug effects , Female , Humans , Integrin beta1/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice, Nude , Phenotype , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Pteridines/pharmacology , Transforming Growth Factor beta/pharmacology , Xenograft Model Antitumor Assays , Polo-Like Kinase 1ABSTRACT
Introduction: Indeterminate pulmonary lesions (IPL) detected by CT pose a significant clinical challenge, frequently necessitating long-term surveillance or biopsy for diagnosis. In this pilot investigation, we performed whole exome sequencing (WES) of plasma cell free (cfDNA) and matched germline DNA in patients with CT-detected pulmonary lesions to determine the feasibility of somatic cfDNA mutations to differentiate benign from malignant pulmonary nodules. Methods: 33 patients with a CT-detected pulmonary lesions were retrospectively enrolled (n = 16 with a benign nodule, n = 17 with a malignant nodule). Following isolation and amplification of plasma cfDNA and matched peripheral blood mononuclear cells (PBMC) from patient blood samples, WES of cfDNA and PBMC DNA was performed. After genomic alignment and filtering, we looked for lung-cancer associated driver mutations and next identified high-confidence somatic variants in both groups. Results: Somatic cfDNA mutations were observed in both groups, with the cancer group demonstrating more variants than the benign group (1083 ± 476 versus 553 ± 519, p < 0.0046). By selecting variants present in >2 cancer patients and not the benign group, we accurately identified 82% (14/17) of cancer patients. Conclusions: This study suggests a potential role for cfDNA for the early identification of lung cancer in patients with CT-detected pulmonary lesions. Importantly, a substantial number of somatic variants in healthy patients with benign pulmonary nodules were also found. Such "benign" variants, while largely unexplored to date, have widespread relevance to all liquid biopsies if cfDNA is to be used accurately for cancer detection.
ABSTRACT
PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. RESULTS: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents. CONCLUSIONS: Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/mTOR pathway inhibitors.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Pyruvate Dehydrogenase Acetyl-Transferring Kinase/metabolism , Receptor, Notch1/genetics , Squamous Cell Carcinoma of Head and Neck/etiology , Squamous Cell Carcinoma of Head and Neck/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Apoptosis/genetics , CRISPR-Cas Systems , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Gene Editing , Gene Expression , Gene Knockdown Techniques , Humans , Loss of Function Mutation , Mice , Protein Kinase Inhibitors/pharmacology , Receptor, Notch1/metabolism , Signal Transduction/drug effects , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/pathologyABSTRACT
PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are currently used in combination with endocrine therapy to treat advanced hormone receptor-positive, HER2-negative breast cancer. Although this treatment doubles time to progression compared with endocrine therapy alone, about 25%-35% of patients do not respond, and almost all patients eventually acquire resistance. Discerning the mechanisms of resistance to CDK4/6 inhibition is crucial in devising alternative treatment strategies. EXPERIMENTAL DESIGN: Palbociclib-resistant cells (MCF-7 and T47D) were generated in a step-wise dose-escalading fashion. Whole-exome sequencing, genome-wide expression analysis, and proteomic analysis were performed in both resistant and parental (sensitive) cells. Pathway alteration was assessed mechanistically and pharmacologically. Biomarkers of altered pathways were examined in tumor samples from patients with palbociclib-treated breast cancer whose disease progressed while on treatment. RESULTS: Palbociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and are also resistant to endocrine therapy (estrogen receptor downregulation). IL6/STAT3 pathway is induced, whereas DNA repair and estrogen receptor pathways are downregulated in the resistant cells. Combined inhibition of STAT3 and PARP significantly increased cell death in the resistant cells. Matched tumor samples from patients with breast cancer who progressed on palbociclib were examined for deregulation of estrogen receptor, DNA repair, and IL6/STAT3 signaling, and results revealed that these pathways are all altered as compared with the pretreatment tumor samples. CONCLUSIONS: Palbociclib resistance induces endocrine resistance, estrogen receptor downregulation, and alteration of IL6/STAT3 and DNA damage response pathways in cell lines and patient samples. Targeting IL6/STAT3 activity and DNA repair deficiency using a specific STAT3 inhibitor combined with a PARP inhibitor could effectively treat acquired resistance to palbociclib.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , DNA Repair/drug effects , Drug Resistance, Neoplasm , Receptors, Estrogen/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Breast Neoplasms/pathology , Cell Line, Tumor , Computational Biology/methods , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Profiling , Humans , Immunohistochemistry , Piperazines/pharmacology , Piperazines/therapeutic use , Protein Kinase Inhibitors/pharmacology , Pyridines/pharmacology , Pyridines/therapeutic use , Treatment OutcomeABSTRACT
PURPOSE: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status.Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. RESULTS: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. CONCLUSIONS: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.
Subject(s)
Antineoplastic Agents/pharmacology , Signal Transduction/drug effects , Squamous Cell Carcinoma of Head and Neck/metabolism , Thiosemicarbazones/pharmacology , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cellular Senescence/drug effects , Cellular Senescence/genetics , Cisplatin/pharmacology , DNA Damage , DNA Replication , Disease Models, Animal , Gene Expression Profiling , Humans , Mice , Protein Binding , Signal Transduction/radiation effects , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/pathology , Stress, Physiological , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor AssaysABSTRACT
Incidence of HPV+ oropharyngeal squamous cell carcinoma (OPSCC) has been increasing dramatically. Although long-term survival rates for these patients are high, they often suffer from permanent radiotherapy-related morbidity. This has prompted the development of de-escalation clinical protocols to reduce morbidity. However, a subset of patients do not respond even to standard therapy and have poor outcomes. It is unclear how to properly identify and treat the high- and low-risk HPV+ OPSCC patients. Since HPV positivity drives radiotherapy sensitivity, we hypothesized that variations in HPV biology may cause differences in treatment response and outcome. By analyzing gene expression data, we identified variations in HPV-related molecules among HPV+ OPSCC. A subset of tumors presented a molecular profile distinct from that of typical HPV+ tumors and exhibited poor treatment response, indicating molecular and clinical similarities with HPV- tumors. These molecular changes were also observed in vitro and correlated with radiation sensitivity. Finally, we developed a prognostic biomarker signature for identification of this subgroup of HPV+ OPSCC and validated it in independent cohorts of oropharyngeal and cervical carcinomas. These findings could translate to improved patient stratification for treatment deintensification and new therapeutic approaches for treatment-resistant HPV-related cancer.
ABSTRACT
Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection.Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status.Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE- tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites.Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727-33. ©2018 AACR.