Molecular jackhammers eradicate cancer cells by vibronic-driven action.

Through the actuation of vibronic modes in cell-membrane-associated aminocyanines, using near-infrared light, a distinct type of molecular mechanical action can be exploited to rapidly kill cells by necrosis. Vibronic-driven action (VDA) is distinct from both photodynamic therapy and photothermal therapy as its mechanical effect on the cell membrane is not abrogated by inhibitors of reactive oxygen species and it does not induce thermal killing. Subpicosecond concerted whole-molecule vibrations of VDA-induced mechanical disruption can be achieved using very low concentrations (500 nM) of aminocyanines or low doses of light (12 J cm-2, 80 mW cm-2 for 2.5 min), resulting in complete eradication of human melanoma cells in vitro. Also, 50% tumour-free efficacy in mouse models for melanoma was achieved. The molecules that destroy cell membranes through VDA have been termed molecular jackhammers because they undergo concerted whole-molecule vibrations. Given that a cell is unlikely to develop resistance to such molecular mechanical forces, molecular jackhammers present an alternative modality for inducing cancer cell death.

TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV-associated oral squamous cell carcinoma.

BACKGROUND: TP53, the most mutated gene in solid cancers, has a profound impact on most hallmarks of cancer. Somatic TP53 mutations occur in high frequencies in head and neck cancers, including oral squamous cell carcinoma (OSCC). Our study aims to understand the role of TP53 gain-of-function mutation in modulating the tumor immune microenvironment (TIME) in OSCC. METHODS: Short hairpin RNA knockdown of mutant p53R172H in syngeneic oral tumors demonstrated changes in tumor growth between immunocompetent and immunodeficient mice. HTG EdgeSeq targeted messenger RNA sequencing was used to analyze cytokine and immune cell markers in tumors with inactivated mutant p53R172H. Flow cytometry and multiplex immunofluorescence (mIF) confirmed the role of mutant p53R172H in the TIME. The gene expression of patients with OSCC was analyzed by CIBERSORT and mIF was used to validate the immune landscape at the protein level. RESULTS: Mutant p53R172H contributes to a cytokine transcriptome network that inhibits the infiltration of cytotoxic CD8+ T cells and promotes intratumoral recruitment of regulatory T cells and M2 macrophages. Moreover, p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Interestingly, p53R172H-mutated tumors are infiltrated with CD8+ and CD4+ T cells expressing programmed cell death protein 1, and these tumors responded to immune checkpoint inhibitor and stimulator of interferon gene 1 agonist therapy. CIBERSORT analysis of human OSCC samples revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our syngeneic mouse tumor model. CONCLUSIONS: These findings demonstrate that syngeneic tumors bearing the TP53R172H gain-of-function mutation modulate the TIME to evade tumor immunity, leading to tumor progression and decreased survival.

Extraforaminal Full-Endoscopic Approach for the Treatment of Lateral Compressive Diseases of the Lumbar Spine.

BACKGROUND: The authors conducted a 2-year retrospective follow-up to investigate the efficiency of an extraforaminal full-endoscopic approach with foraminoplasty used to treat lateral compressive diseases of the lumbar spine in 247 patients. METHODS: The visual analogue scale (VAS), Oswestry disability index (ODI), and MacNab scale were used to analyze the results collected during the preoperative and postoperative periods. RESULTS: The most common diagnosis was disk herniation with lateral recess stenosis, and the most common surgical level among patients was between L4 and L5 on the left side. Pain decreased over time, as determined during sessions held to evaluate pain in the lumbar, gluteal, led, and foot regions. The ODI demonstrated significant enhancement over the evaluation period and the MacNab scale classified the surgery as good or excellent. The most common complication was dysesthesia. CONCLUSIONS: An extraforaminal full-endoscopic approach with foraminoplasty can be recommended in cases of lateral herniation or stenosis for patients with symptoms of radiculopathy, and for those who have not responded to conventional rehabilitation treatment or chronic pain management. Few complications arose as a result of this approach, and most of them were treated clinically.

Sagittal Plane Geometry of Cervical, Thoracic, and Lumbar Endplates.

BACKGROUND: Many studies have emphasized the importance of interface contact between implants and the vertebral endplate (VE). The goal of this study was to analyze the shape and other specific parameters of the VE to provide reference data for better implant interface contact in intervertebral disc space procedures. METHODS: Cervical, thoracic, and lumbar spine midsagittal plane magnetic resonance images of 100 adults (58 women) were analyzed. The morphology of the VEs was classified as concave, convex, flat, or irregular. Midsagittal endplate length (ML), endplate concavity depth (ECD), and endplate concavity axis (ECA) location were measured in the midsagittal plane. The parameters were compared between the cervical, thoracic, and lumbar spines and between the sexes. RESULTS: The VE morphology, ML, ECD, and ECA showed variations along the spine, mainly in the cervical and lower lumbar spines. The sagittal geometry of the VE was not flat or uniform along the cervical, thoracic, and lumbar spines. Different morphological types were observed along different spinal segments and according to sex. In the cervical spine, the majority of cranial VEs were flat, while caudal VEs were mostly concave. CONCLUSION: Sagittal VE geometry should be taken into consideration during the use of intervertebral cages or disc arthroplasty.

Mutant p53 drives an immune cold tumor immune microenvironment in oral squamous cell carcinoma.

The critical role of the tumor immune microenvironment (TIME) in determining response to immune checkpoint inhibitor (ICI) therapy underscores the importance of understanding cancer cell-intrinsic mechanisms driving immune-excluded ("cold") TIMEs. One such cold tumor is oral cavity squamous cell carcinoma (OSCC), a tobacco-associated cancer with mutations in the TP53 gene which responds poorly to ICI therapy. Because altered TP53 function promotes tumor progression and plays a potential role in TIME modulation, here we developed a syngeneic OSCC models with defined Trp53 (p53) mutations and characterized their TIMEs and degree of ICI responsiveness. We observed that a carcinogen-induced p53 mutation promoted a cold TIME enriched with immunosuppressive M2 macrophages highly resistant to ICI therapy. p53-mutated cold tumors failed to respond to combination ICI treatment; however, the combination of a programmed cell death protein 1 (PD-1) inhibitor and stimulator of interferon genes (STING) agonist restored responsiveness. These syngeneic OSCC models can be used to gain insights into tumor cell-intrinsic drivers of immune resistance and to develop effective immunotherapeutic approaches for OSCC and other ICI-resistant solid tumors.

Ubiquitin of Entamoeba histolytica induces antibody response in patients with invasive amoebiasis.

Entamoeba histolytica causes amoebic liver abscess (ALA) in humans. The injury of target cells by E. histolytica includes processes controlled by the ubiquitin Ehub. Previously, we found immunodominance of Ehub glycan moieties using immunized rabbits. In this work, we analysed dominance of antibodies to the glycoprotein Ehub in the sera from 52 patients with ALA. Controls were sera from 20 healthy people living in endemic areas with a high seroprevalence of antibodies to amoebas, and 20 patients with alcoholic hepatitis (AH) to rule out the cross-reaction of Ehub with autoantibodies induced by liver damage. Antigens were trophozoite extract, glycoprotein Ehub and the recombinant protein E. histolytica recombinant ubiquitin (rEhub). The sera from healthy volunteers and patients with AH do not have antibodies to glycoprotein Ehub. Surprisingly, only the antibodies from patients with ALA recognized the glycoprotein Ehub, and some sera gave a faint reaction with the recombinant protein, especially because evolutionarily, the ubiquitin is conserved between species. This is the first report demonstrating that antibodies to ubiquitin Ehub are induced exclusively in patients with invasive amoebiasis, and the antibody response is mainly to the glycoprotein, indicating glycans are immunodominant. Inhibitors of the Ehub glycans could be potential treatment for amoebiasis by selectively damaging trophozoites.

Dural Injury Treatment with a Full-Endoscopic Transforaminal Approach: A Case Report and Description of Surgical Technique.

Introduction: The objective of this study was to describe a surgical technique that uses transforaminal full-endoscopic access, which is different from the existing protocol, and to demonstrate another method of dural tear repair during endoscopic spine surgery. Background: Endoscopic spine surgery was initially described for lumbar disc pathologies. Technical advances and new materials have made it possible to treat cervical and thoracic spinal degenerative disorders. These advances have also made it possible to treat surgical complications, notably dural tears with CSF fistulas. The literature indicates that the incidence of these injuries ranges from 1% to 17%. Materials and Methods: Descriptive technical note of innovative and improved endoscopic surgical procedure exemplified with illustrative clinical case and comparative literature review. Results: There is only one report describing a full-endoscopic suture technique for dural sac repair. The gold standard for treatment of the most significant nonpunctate lesions continues to be a conversion to open surgery for lesion closure. Conversion can be problematic because most surgeries are performed under sedation and local anesthesia. Conclusions: In this case report and the new endoscopic suture technique described here, we show that primary correction of dural tears through endoscopy is possible. In addition to representing a paradigm break in solving one of the main complications of these procedures, it can expand the possibilities of spine endoscopy.

Genetic Changes Driving Immunosuppressive Microenvironments in Oral Premalignancy.

Oral premalignant lesions (OPLs) are the precursors to oral cavity cancers, and have variable rates of progression to invasive disease. As an intermediate state, OPLs have acquired a subset of the genomic alterations while arising in an oral inflammatory environment. These specific genomic changes may facilitate the transition to an immune microenvironment that permits malignant transformation. Here, we will discuss mechanisms by which OPLs develop an immunosuppressive microenvironment that facilitates progression to invasive cancer. We will describe how genomic alterations and immune microenvironmental changes co-evolve and cooperate to promote OSCC progression. Finally, we will describe how these immune microenvironmental changes provide specific and unique evolutionary vulnerabilities for targeted therapies. Therefore, understanding the genomic changes that drive immunosuppressive microenvironments may eventually translate into novel biomarker and/or therapeutic approaches to limit the progression of OPLs to potential lethal oral cancers.

Monoclonal Antibodies Generation: Updates and Protocols on Hybridoma Technology.

Since its inception in 1975, the hybridoma technology revolutionized science and medicine, facilitating discoveries in almost any field from the laboratory to the clinic. Many technological advancements have been developed since then, to create these "magical bullets." Phage and yeast display libraries expressing the variable heavy and light domains of antibodies, single B-cell cloning from immunized animals of different species including humans or in silico approaches, all have rendered a myriad of newly developed antibodies or improved design of existing ones. However, still the majority of these antibodies or their recombinant versions are from hybridoma origin, a preferred methodology that trespass species barriers, due to the preservation of the natural functions of immune cells in producing the humoral response: antigen specific immunoglobulins. Remarkably, this methodology can be reproduced in small laboratories without the need of sophisticate equipment. In this chapter, we will describe the most recent methods utilized by our Monoclonal Antibodies Core Facility at the University of Texas-M.D. Anderson Cancer Center. During the last 10 years, the methods, techniques, and expertise implemented in our core had generated more than 350 antibodies for various applications.

Sleeping Beauty Transposon Mutagenesis Identifies Genes Driving the Initiation and Metastasis of Uterine Leiomyosarcoma.

Uterine leiomyosarcoma (ULMS) is a malignancy, which arises from the uterine smooth muscle. Because of its rarity, aggressive nature, and extremely poor prognosis, the molecular mechanisms driving ULMS remain elusive. To identify candidate cancer genes (CCG) driving ULMS, we conducted an in vivo Sleeping Beauty (SB) transposon mutagenesis screen in uterine myometrium-specific, PTEN knockout, KRAS mutant (PTEN KO/KRAS) mice. ULMS quickly developed in SB PTEN KO/KRAS mice, but not in PTEN KO/KRAS mice, demonstrating the critical importance of SB mutagenesis for driving ULMS in this model. Subsequent sequencing of SB insertion sites in these tumors identified 19 ULMS CCGs that were significantly enriched in known cancer genes. Among them, Zfp217 and Sfmbt2 functioned at early stages of tumor initiation and appeared to be oncogenes. Expression of ZNF217, the human homolog of ZFP217, was shown to be elevated in human ULMS compared with paired normal uterine smooth muscle, where it negatively correlated with patient prognosis. Inhibition of ZNF217 suppressed, whereas overexpression induced, proliferation, survival, migration, and stemness of human ULMS. In a second ex vivo ULMS SB metastasis screen, three CCGs were identified that may drive ULMS metastasis to the lung. One of these CCGs, Nrd1 (NRDC in humans), showed stronger expression in human metastatic tumors compared with primary ULMS and negatively associated with patient survival. NRDC knockdown impaired migration and adhesion without affecting cell proliferation, whereas overexpression had the opposite effect. Together, these results reveal novel mechanism driving ULMS tumorigenesis and metastasis and identify ZNF217 and NRDC as potential targets for ULMS therapy. SIGNIFICANCE: An in vivo Sleeping Beauty transposon mutagenesis screen identifies candidate cancer genes that drive initiation and progression of uterine leiomyosarcoma and may serve as therapeutic targets.

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression.

The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.

Low doses of methylnaltrexone inhibits head and neck squamous cell carcinoma growth in vitro and in vivo by acting on the mu-opioid receptor.

The Mu-opioid receptor (MOR) has been implicated in tumorigenesis and metastasis. Methylnaltrexone (MNTX), an antagonist of MOR, has shown to inhibit tumor growth and metastasis in lung cancer cell lines. The effect of MNTX on other cell lines such as head and neck squamous cell carcinoma (HNSCC) has not been investigated. We measured the expression and activity of the receptor in different HNSCC cell lines. Then, we evaluated the impact of modulating the expression MOR and the effect of MNTX on the proliferation, clonogenic activity, invasion, and migration of two HNSCC (FaDu and MDA686Tu) cell lines expressing MOR and one cell line (UMSCC47) not expressing the receptor. We also evaluated the impact of MNTX on tumor growth and metastasis formation in vivo. Activation of the receptor with [d-Ala2,N-Me-Phe4, Gly5-ol] (DAMGO) caused a significant reduction in cyclic adenosine monophosphate levels in FaDu cells. Knockdown of MOR inhibited in vitro aggressive cell behaviors on FaDu and MDA686Tu cells and correlated with a reduction in markers of epithelial-mesenchymal transition. In vitro studies showed that MNTX strongly inhibited the proliferation, clonogenic activity, invasion, and migration of FaDu and MDA686Tu cells but has no effect on UMSCC47 cells. In vivo experiments demonstrated that MNTX suppresses tumor growth in HNSCC cell tumor-bearing mice. Our studies indicate that MOR could be considered as a therapeutic target to treat HNSCC.

Local Anti-PD-1 Delivery Prevents Progression of Premalignant Lesions in a 4NQO-Oral Carcinogenesis Mouse Model.

Although the principle of systemic treatment to prevent the progression of oral premalignant lesions (OPL) has been demonstrated, there remains a lack of consensus about an optimal approach that balances clinical efficacy with toxicity concerns. Recent advances in cancer therapy using approaches targeting the tumor immune microenvironment (TIME) including immune-checkpoint inhibitors indicate that these agents have significant clinically activity against different types of cancers, including oral cancer, and therefore they may provide an effective oral cancer prevention strategy for patients with OPLs. Our past work showed that systemic delivery of a monoclonal antibody to the programmed death receptor 1 (PD-1) immune checkpoint can inhibit the progression of OPLs to oral cancer in a syngeneic murine oral carcinogenesis model. Here we report a novel approach of local delivery of a PD-1 immune-checkpoint inhibitor loaded using a hydrogel, which significantly reduces the progression of OPLs to carcinomas. In addition, we detected a significant infiltration of regulatory T cells associated with oral lesions with p53 mutation, and a severe loss of expression of STING, which correlated with a decreased infiltration of dendritic cells in the oral lesions. However, a single local dose of PD-1 inhibitor was found to restore stimulator of interferon response cGAMP interactor 1 (STING) and CD11c expression and increase the infiltration of CD8+ T cells into the TIME irrespective of the p53 mutational status. Overall, we provide evidence for the potential clinical value of local delivery of biomaterials loaded with anti-PD-1 antibodies to prevent malignant progression of OPLs. PREVENTION RELEVANCE: Oral cancer is an aggressive disease, with an overall survival rate of 50%. Preinvasive histologic abnormalities such as tongue dysplasia represent an early stage of oral cancer; however, there are no treatments to prevent oral carcinoma progression. Here, we combined biomaterials loaded with an immunotherapeutic agent preventing oral cancer progression.

Surgery for lumbar disc herniation: open x minimally invasive technique / Cirurgia de hérnia discal lombar: técnica aberta x minimamente invasiva / Cirugía de hernia discal lumbar: técnica abierta x mínimamente invasiva

ABSTRACT Objective: In Brazil, there are no studies comparing endoscopic treatment of lumbar disc herniation with the conventional open technique in SUS (Unified Health System) with regard to hospitalization time and complications occurring within one year, which is the objective of this study. Methods: A survey of 32 surgeries performed in 2019 (11 open and 21 endoscopic) to evaluate pain parameters before and after surgery (VAS), days of hospitalization, and complications. The data were submitted to statistical analysis (ANOVA) using the Kruskal-Wallis test. Results: Fourteen patients were female and eighteen were male, with a mean age of 41.35 years (p> 0.05 between sexes). The pre- and postoperative VAS for pain radiating to the lower limb were similar between the groups: 8.5 ± 0.82 with the open technique and 8.19 ± 1.15 with endoscopic technique. In both groups there was an improvement in the pain pattern with a significant reduction in the VAS (p < 0.05) and there was no statistical relevance between the groups in terms of pain improvement. There was statistical relevance between the groups in the comparison of days of hospitalization required, with the group submitted to endoscopic surgery having a lower number of days. The complications reported were compatible with those found in the literature (postoperative dysesthesia, new herniation). Conclusions: The endoscopic technique resulted in an important reduction in the number of days of hospitalization, a factor with a high impact on the costs of any surgical procedure, which can be a determining factor in the feasibility of minimally invasive techniques. Level of evidence IV; Therapeutic Study.

RESUMO Objetivos: No Brasil, não há estudos que comparem o tratamento endoscópico de hérnia de disco lombar no SUS (Sistema Único de Saúde) com a técnica aberta convencional, no que diz respeito aos resultados com relação ao tempo de internação e complicações ocorridas em um ano, o que vem a ser o objetivo deste estudo. Métodos: Levantamento de 32 cirurgias realizadas em 2019 (11 por via aberta e 21 por via endoscópica) para avaliar os parâmetros de dor antes e depois da cirurgia (EVA), dias de internação e complicações. Os dados foram submetidos à análise estatística (ANOVA) com o teste de Kruskal-Wallis. Resultados: Catorze pacientes eram do sexo feminino e 18 do sexo masculino, com média de idade de 41,35 anos (p > 0,05 para os dois sexos). A EVA de dor irradiada para o membro inferior no pré e pós-operatório foi semelhante entre os grupos: 8,5 ± 0,82 com a técnica aberta e 8,19 ± 1,15 com a técnica endoscópica. Em ambos os grupos houve melhora do padrão de dor com redução significativa da EVA (p < 0,05) e não houve relevância estatística entre os grupos quanto à melhora do dor. Na comparação das diárias de internação necessárias houve relevância estatística entre os grupos, sendo que o grupo submetido à endoscopia teve número menor de diárias. As complicações relatadas são compatíveis com as encontradas na literatura (disestesia pós-operatória, nova herniação). Conclusões: A técnica endoscópica resultou em redução importante do número de dias de internação, fator com alto impacto nos custos de qualquer procedimento cirúrgico, que pode ser determinante para viabilizar técnicas minimamente invasivas. Nível de evidência IV; Estudo Terapêutico.

RESUMEN Objetivos: En Brasil, no hay estudios que comparen el tratamiento endoscópico de hernia de disco lumbar en el SUS (Sistema Único de Salud) con la técnica abierta convencional, en lo que refiere a los resultados con relación al tiempo de internación y complicaciones ocurridas en un año, lo que viene a ser el objetivo de este estudio. Métodos: Levantamiento de 32 cirugías realizadas en 2019 (once por vía abierta y veintiuna por vía endoscópica) para evaluar los parámetros de dolor antes y después de la cirugía (EVA), días de internación y complicaciones. Los datos fueron sometidos a análisis estadístico (ANOVA) con el test de Kruskal-Wallis. Resultados: Catorce pacientes eran del sexo femenino y dieciocho del sexo masculino con promedio de edad de 41,35 años (p>0,05 para los dos sexos). La EVA de dolor irradiado para el miembro inferior en el pre y postoperatorio fue semejante entre los grupos: 8,5±0,82 con la técnica abierta y 8,19±1,15 con la técnica endoscópica. En ambos grupos hubo mejoras del patrón de dolor con reducción significativa de la EVA (p<0,05) y no hubo relevancia estadística entre los grupos cuanto a la mejora del dolor. En la comparación de los días de internación necesarios hubo relevancia estadística entre los grupos, siendo que el grupo sometido a la endoscopia tuvo número menor de días de internación. Las complicaciones relatadas son compatibles con las encontradas en la literatura (disestesia postoperatoria, nueva herniación). Conclusiones: La técnica endoscópica resultó en reducción importante del número de días de internación, factor con alto impacto en los costos de cualquier procedimiento quirúrgico, que puede ser determinante para viabilizar técnicas mínimamente invasivas. Nivel de evidencia IV; Estudio Terapéutico.

Glycan moieties in Entamoeba histolytica ubiquitin are immunodominant.

The ubiquitin-proteasome system plays a central role performing several functions to maintain parasite homeostasis. We have reported the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin (EhUb). Here we examined the immunogenicity and antigenicity of carbohydrates in EhUbiquitin. Rabbits were immunized with purified EhUbiquitin or purified recombinant rUb expressed by E. coli. Using Western Blot, we explored the immunogenicity and antigenicity of protein portion and carbohydrates moiety. Interestingly, immunized rabbits produced antibodies to both Ub glycoprotein and rUb; but antibodies against carbohydrates were immunodominant, rather than antibodies to the protein moiety of EhUbiquitin. In addition, we observed that antibodies to protein moiety are not conserved in serum unless antigen is continually administrated. Conversely, anti-Ub glycoprotein antibodies are well maintained in circulation. In humans, infection with Entamoeba histolytica induces strong IgG anti-Ub response. The human antibodies recognize both, the protein moieties and the glycosylated structure. Entamoeba histolytica ubiquitin is immunogenic and antigenic. The glycan moieties are immunodominant and induces IgG. These data open the door to use carbohydrates as potential targets for diagnose tests, drugs and vaccine to prevent this parasitic disease.

Characterization of N-glycosylations in Entamoeba histolytica ubiquitin.

Entamoeba histolytica harbors an extensive intracellular distribution of ubiquitin-proteasome systems important for numerous cellular processes. However, glycosylation studies of ubiquitin-proteasome components have not yet been elucidated. Here we report the partial characterization of N-linked glycosylation profile in E. histolytica ubiquitin by Fluorophore-Assisted Carbohydrate Electrophoresis (FACE), Nanoelectrospray Ionization-Tandem Mass Spectrometry (NSI-MS), Matrix-Assisted Laser-Desorption time-of-flight Mass Spectrometry (MALDI-TOF MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. To our knowledge, the data presented in this report represents the first structural glycomics analysis of E. histolytica ubiquitin, while most of the reports are performed on whole parasitic glycan profiles. The glycan profile of E. histolytica ubiquitin has high mannose N-glycan structures. The N-linked glycan profile showed fragments from Hex3HexNAc2 to Hex9HexNAc2. Based in our findings and ubiquitin function, we hypothesize that the same ubiquitin Asn-Asp-Ser sequon carries heterogenic glycosylations, at different metabolic pathway stages according to ubiquitin functional requirements. Finally, we propose a set of possible high mannose N-glycan structures that will help to elucidate the ubiquitin biochemical composition and may well represent good targets for anti-amoebic drugs.

In vivo loss-of-function screens identify KPNB1 as a new druggable oncogene in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has not been changed significantly during several decades. To seek new therapeutic targets for EOC, we performed an in vivo dropout screen in human tumor xenografts using a pooled shRNA library targeting thousands of druggable genes. Then, in follow-up studies, we performed a second screen using a genome-wide CRISPR/Cas9 library. These screens identified 10 high-confidence drug targets that included well-known oncogenes such as ERBB2 and RAF1, and novel oncogenes, notably KPNB1, which we investigated further. Genetic and pharmacological inhibition showed that KPNB1 exerts its antitumor effects through multiphase cell cycle arrest and apoptosis induction. Mechanistically, proteomic studies revealed that KPNB1 acts as a master regulator of cell cycle-related proteins, including p21, p27, and APC/C. Clinically, EOC patients with higher expression levels of KPNB1 showed earlier recurrence and worse prognosis than those with lower expression levels of KPNB1. Interestingly, ivermectin, a Food and Drug Administration-approved antiparasitic drug, showed KPNB1-dependent antitumor effects on EOC, serving as an alternative therapeutic toward EOC patients through drug repositioning. Last, we found that the combination of ivermectin and paclitaxel produces a stronger antitumor effect on EOC both in vitro and in vivo than either drug alone. Our studies have thus identified a combinatorial therapy for EOC, in addition to a plethora of potential drug targets.

Identification of New Tumor Suppressor Genes in Triple-Negative Breast Cancer.

Although genomic sequencing has provided a better understating of the genetic landmarks in triple-negative breast cancer (TNBC), functional validation of candidate cancer genes (CCG) remains unsolved. In this study, we used a transposon mutagenesis strategy based on a two-step sleeping beauty (SB) forward genetic screen to identify and validate new tumor suppressors (TS) in this disease. We generated 120 siRNAs targeting 40 SB-identified candidate breast cancer TS genes and used them to downregulate expression of these genes in four human TNBC cell lines. Among CCG, whose SB-mediated genetic mutation resulted in increased cellular proliferation in all cell lines tested, the genes ADNP, AP2B1, TOMM70A, and ZNF326 showed TS activity in tumor xenograft studies. Subsequent studies showed that ZNF326 regulated expression of multiple epithelial-mesenchymal transition and cancer stem cell (CSC) pathway genes. It also modulated expression of TS genes involved in the regulation of migration and cellular invasion and was a direct transcriptional activator of genes that regulate CSC self-renewal. ZNF326 expression associated with TNBC patient survival, with ZNF326 protein levels showing a marked reduction in TNBC. Our validation of several new TS genes in TNBC demonstrate the utility of two-step forward genetic screens in mice and offer an invaluable tool to identify novel candidate therapeutic pathways and targets. Cancer Res; 77(15); 4089-101. ©2017 AACR.

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression.

Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1 Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.