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Glioblastoma multiform (GBM) is the most frequent primitive brain tumor with a high recurrence and mortality. Histone deacetylase inhibitors (HDACi) have evoked great interest because they are able to change transcriptomic profiles to promote tumor cell death but also induce side effects due to the lack of selectivity. We show in this paper new anticancer properties and mechanisms of action of low concentrations of vorinostat on various GBM cells which acts by affecting microtubule cytoskeleton in a non-histone 3 (H3) manner. Indeed, vorinostat induces tubulin acetylation and detyrosination, affects EB stabilizing cap on microtubule plus ends and suppresses microtubule dynamic instability. We previously identified EB1 overexpression as a marker of bad prognostic in GBM. Interestingly, we show for the first time to our knowledge, a strong decrease of EB1 expression in GBM cells by a drug. Altogether, our results suggest that low dose vorinostat, which is more selective for HDAC6 inhibition, could therefore represent an interesting therapeutic option for GBM especially in patients with EB1 overexpressing tumor with lower expected side effects. A validation of our hypothesis is needed during future clinical trials with this drug in GBM.

Objectives This study analyzed an artificial intelligence (AI) deep learning method with a three-dimensional deep convolutional neural network (3D DCNN) in regard to diagnostic accuracy to differentiate malignant pleural mesothelioma (MPM) from benign pleural disease using FDG-PET/CT results. Results For protocol A, the area under the ROC curve (AUC)/sensitivity/specificity/accuracy values were 0.825/77.9% (81/104)/76.4% (55/72)/77.3% (136/176), while those for protocol B were 0.854/80.8% (84/104)/77.8% (56/72)/79.5% (140/176), for protocol C were 0.881/85.6% (89/104)/75.0% (54/72)/81.3% (143/176), and for protocol D were 0.896/88.5% (92/104)/73.6% (53/72)/82.4% (145/176). Protocol D showed significantly better diagnostic performance as compared to A, B, and C in ROC analysis (p = 0.031, p = 0.0020, p = 0.041, respectively). Materials and methods Eight hundred seventy-five consecutive patients with histologically proven or suspected MPM, shown by history, physical examination findings, and chest CT results, who underwent FDG-PET/CT examinations between 2007 and 2017 were investigated in a retrospective manner. There were 525 patients (314 MPM, 211 benign pleural disease) in the deep learning training set, 174 (102 MPM, 72 benign pleural disease) in the validation set, and 176 (104 MPM, 72 benign pleural disease) in the test set. Using AI with PET/CT alone (protocol A), human visual reading (protocol B), a quantitative method that incorporated maximum standardized uptake value (SUVmax) (protocol C), and a combination of PET/CT, SUVmax, gender, and age (protocol D), obtained data were subjected to ROC curve analyses. Conclusions Deep learning with 3D DCNN in combination with FDG-PET/CT imaging results as well as clinical features comprise a novel potential tool shows flexibility for differential diagnosis of MPM.

Further characterization of thymic epithelial tumors (TETs) is needed. Genomic information from 102 evaluable TETs from The Cancer Genome Atlas (TCGA) dataset and from the IU-TAB-1 cell line (type AB thymoma) underwent clustering analysis to identify molecular subtypes of TETs. Six novel molecular subtypes (TH1-TH6) of TETs from the TCGA were identified, and there was no association with WHO histologic subtype. The IU-TAB-1 cell line clustered into the TH4 molecular subtype and in vitro testing of candidate therapeutics was performed. The IU-TAB-1 cell line was noted to be resistant to everolimus (mTORC1 inhibitor) and sensitive to nelfinavir (AKT1 inhibitor) across the endpoints measured. Sensitivity to nelfinavir was due to the IU-TAB-1 cell line’s gain-of function (GOF) mutation in PIK3CA and amplification of genes observed from array comparative genomic hybridization (aCGH), including AURKA, ERBB2, KIT, PDGFRA and PDGFB, that are known upregulate AKT, while resistance to everolimus was primarily driven by upregulation of downstream signaling of KIT, PDGFRA and PDGFB in the presence of mTORC1 inhibition. We present a novel molecular classification of TETs independent of WHO histologic subtype, which may be used for preclinical validation studies of potential candidate therapeutics of interest for this rare disease.

Xeroderma Pigmentosum (XP) is a rare genetic disorder with a poor prognosis due to high photosensitivity in affected patients. Herein, we describe the first case of the use of cemiplimab in a patient with XP, a 19-year-old girl presented with locally advanced squamous cell carcinoma of the right periorbital and nasal region. This treatment has been undertaken after a cycle of proton beam radiotherapy. Besides, it is reported a description of the few cases in the literature describing the effectiveness of immunotherapy on skin cancers in XP-patients. This case is in line with those reported, underlining how anti-PD1 monoclonal antibodies may be a promising treatment in this genodermatosis.

Cutaneous apocrine carcinoma is an extreme rare malignancy derived from a sweat gland. Histologically sweat gland cancers resemble metastatic mammary apocrine carcinomas, but the genetic landscape remains poorly understood. Here, we report a rare metastatic case with a PALB2 aberration identified previously as a familial susceptibility gene for breast cancer in the Finnish population. As PALB2 exhibits functions in the BRCA1/2-RAD51-dependent homologous DNA recombination repair pathway, we sought to use ex vivo functional screening to explore sensitivity of the tumor cells to therapeutic targeting of DNA repair. Drug screening suggested sensitivity of the PALB2 deficient cells to BET-bromodomain inhibition, and modest sensitivity to DNA-PKi, ATRi, WEE1i and PARPi. A phenotypic RNAi screen of 300 DNA repair genes was undertaken to assess DNA repair targeting in more detail. Core members of the HR and MMEJ pathways were identified to be essential for viability of the cells. RNAi inhibition of RAD52-dependent HR on the other hand potentiated the efficacy of a novel BETi ODM-207. Together these results describe the first ever CAC case with a BRCAness genetic background, evaluate combinatorial DNA repair targeting, and provide a data resource for further analyses of DNA repair targeting in PALB2 deficient cancers.

Non-invasive clinical diagnostics of bladder cancer is feasible via a set of chemically distinct molecules including macromolecular tumor markers such as polypeptides and nucleic acids. In terms of tumor-related aberrant gene expression, RNA transcripts are the primary indicator of tumor-specific gene expression as for polypeptides and their metabolic products occur subsequently. Thus, in case of bladder cancer, urine RNA represents an early potentially useful diagnostic marker. Here we describe a systematic deep transcriptome analysis of representative pools of urine RNA collected from healthy donors versus bladder cancer patients according to established SOPs. This analysis revealed RNA marker candidates reflecting coding sequences, non-coding sequences, and circular RNAs. Next, we designed and validated PCR amplicons for a set of novel marker candidates and tested them in human bladder cancer cell lines. We identified linear and circular transcripts of the S100 Calcium Binding Protein 6 (S100A6) and translocation associated membrane protein 1 (TRAM1) as highly promising potential tumor markers. This work strongly suggests exploiting urine RNAs as diagnostic markers of bladder cancer and it suggests specific novel markers. Further, this study describes an entry into the tumor-biology of bladder cancer and the development of gene-targeted therapeutic drugs.

Retinoblastoma is the most common pediatric intraocular malignant tumor. While retinoblastoma initiation is triggered by the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, tumor progression requires additional epigenetic changes, retinoblastoma genomes being quite stable. Although the management of RB has recently improved, new therapeutic agents are necessary to improve the treatment of advanced forms of retinoblastoma. In this report, we analyzed the pro-death effect of piperlongumine (PL), a natural compound isolated from Piper longum L., on two human retinoblastoma cell lines, WERI-Rb and Y79. The effects of PL on cell proliferation, cell death and cell cycle were investigated. PL effectively inhibited cell growth, impacted the cell cycle by decreasing the level of cyclins and CDK1 and increasing CDKN1A and triggered a caspase-3 independant cell death process in which reactive oxygen species (ROS) production is a major player. Indeed, PL toxicity in retinoblastoma cell lines was inhibited by a ROS scavenger N-acetyl-l-cysteine (NAC) treatment. These findings suggest that PL reduces tumor growth and induces cell death by regulating the cell cycle.

Hypoxia stimulates neoangiogenesis, promoting tumor outgrowth, and triggers the epithelial-mesenchymal transition (EMT), which bestows cells with mesenchymal traits and multi-lineage differentiation potential. Here, we investigated whether EMT can confer endothelial attributes upon carcinoma cells, augmenting tumor growth and vascularization. Following orthotopic implantation of MCF-7 human epithelial breast cancer cells into mice, tumors of different sizes were immunostained for markers of hypoxia and EMT. Larger tumors were well-vascularized with CD31-positive cells of human origin. Hypoxic regions, demarcated by HIF-1α staining, exhibited focal areas of E-cadherin loss and elevated levels of vimentin and the EMT-mediator FOXC2. Implantation of MCF-7 cells, co-mixed with human mammary epithelial (HMLE) cells overexpressing the EMT-inducer Snail, markedly potentiated tumor growth and vascularization, compared with MCF-7 cells injected alone or co-mixed with HMLE-vector cells. Intra-tumoral vessels contained CD31-positive cells derived from either donor cell type. FOXC2 knockdown abrogated the potentiating effects of HMLE-Snail cells on MCF-7 tumor growth and vascularization, and compromised endothelial transdifferentiation of mesenchymal cells cultured in endothelial growth medium. Hence, cells that have undergone EMT can promote tumor growth and neovascularization either indirectly, by promoting endothelial transdifferentiation of carcinoma cells, or directly, by acquiring an endothelial phenotype, with FOXC2 playing key roles in these processes.

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches.

Purpose: The purpose of this study is to identify novel urine protein biomarkers of bladder cancer using a Luminex based screening platform. Materials and Methods: The current study examines urine samples from 66 subjects, comprised of 31 Urology clinic controls and 35 bladder cancer patients, using a Luminex based screening platform. ELISA validation was carried out for the top 4 prospective urine biomarkers using an independent cohort of 20 Urology clinic controls and 60 bladder cancer (BC) subjects. Results: Of the 16 proteins screened by Luminex, 10 showed significant elevation in BC compared to the controls. Eight of these urine proteins were able to differentiate BC from control urine with ROC AUC values exceeding 0.70 at p < 0.0001, with specificity values exceeding 0.9. Upon ELISA validation, urine IL-1α, IL-1ra, and IL-8 were able to distinguish control urine from urine drawn from various bladder cancer stages, with IL-8 being the best discriminator. Compared to members of the IL-1 cytokine family, urine IL-8 was also best at discriminating T1 and/or T2–T4 from Ta BC (ROC AUC ≥ 0.83), as well as high grade from low grade BC (ROC AUC ≥ 0.82). Conclusions: These findings suggest that urine IL-1α, IL-1ra and IL-8 are useful indicators of bladder cancer. Urine IL-8 not only distinguishes bladder cancer from controls, it also discriminates high grade from low grade disease, and the successive clinical stages of bladder cancer. While supportive of previous reports, these findings warrant further analysis in prospective cohorts.

Somatic mutation signatures are an informative facet of cancer aetiology, however they are rarely useful for predicting patient outcome. The aim of this study is to evaluate the utility of a panel of 142 mutation-signature–associated metrics (P142) for predicting cancer progression in patients from a ‘TCGA PanCancer Atlas’ cohort. The P142 metrics are comprised of AID/APOBEC and ADAR deaminase associated SNVs analyzed for codon context, strand bias, and transitions/transversions. TCGA tumor-normal mutation data was obtained for 10,437 patients, representing 31 of the most prevalent forms of cancer. Stratified random sampling was used to split patients into training, tuning and validation cohorts for each cancer type. Cancer specific machine learning (XGBoost) models were built using the output from the P142 panel to predict patient Progression Free Survival (PFS) status as either “High PFS” or “Low PFS”. Predictive performance of each model was evaluated using the validation cohort. Models accurately predicted PFS status for several cancer types, including adrenocortical carcinoma, glioma, mesothelioma, and sarcoma. In conclusion, the P142 panel of metrics successfully predicted cancer progression status in patients with some, but not all cancer types analyzed. These results pave the way for future studies on cancer progression associated signatures.

Approximately 15% of colorectal cancer (CRC) cases present with high levels of microsatellite instability (MSI-H). Bulk RNA-sequencing approaches have been employed to elucidate transcriptional differences between MSI-H and microsatellite stable (MSS) CRC tumors. These approaches are frequently confounded by the complex cellular heterogeneity of tumors. We performed single-cell deconvolution of bulk RNA-sequencing on The Cancer Genome Atlas colon adenocarcinoma (TCGA-COAD) dataset. Cell composition within each dataset was estimated using CIBERSORTx. Cell composition differences were analyzed using linear regression. Significant differences in abundance were observed for 13 of 19 cell types between MSI-H and MSS/MSI-L tumors in TCGA-COAD. This included a novel finding of increased enteroendocrine (q = 3.71E-06) and reduced colonocyte populations (q = 2.21E-03) in MSI-H versus MSS/MSI-L tumors. We were able to validate some of these differences in an independent biopsy dataset. By incorporating cell composition into our regression model, we identified 3,193 differentially expressed genes (q = 0.05), of which 556 were deemed novel. We subsequently validated many of these genes in an independent dataset of colon cancer cell lines. In summary, we show that some of the challenges associated with cellular heterogeneity can be overcome using single-cell deconvolution, and through our analysis we highlight several novel gene targets for further investigation.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal type of cancer. In this study, we undertook a pairwise comparison of gene expression pattern between tumor tissue and its matching adjacent normal tissue for 45 PDAC patients and identified 22 upregulated and 32 downregulated genes. PPI network revealed that fibronectin 1 and serpin peptidase inhibitor B5 were the most interconnected upregulated-nodes. Virtual screening identified bleomycin exhibited reasonably strong binding to both proteins. Effect of bleomycin on cell viability was examined against two PDAC cell lines, AsPC-1 and MIA PaCa-2. AsPC-1 did not respond to bleomycin, however, MIA PaCa-2 responded to bleomycin with an IC50 of 2.6 μM. This implicates that bleomycin could be repurposed for the treatment of PDAC, especially in combination with other chemotherapy agents. In vivo mouse xenograft studies and patient clinical trials are warranted to understand the functional mechanism of bleomycin towards PDAC and optimize its therapeutic efficacy. Furthermore, we will evaluate the antitumor activity of the other identified drugs in our future studies.

Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.

Around 3–7% of patients with non-small cell lung cancer (NSCLC), which represent 85% of diagnosed lung cancers, have a rearrangement in the ALK gene that produces an abnormal activity of the ALK protein cell signaling pathway. The developed ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, ceritinib, alectinib, brigatinib and lorlatinb present good performance treating ALK+ NSCLC, although all patients invariably develop resistance due to ALK secondary mutations or bypass mechanisms. In the present study, we compare the potential differences between brigatinib and alectinib’s mechanisms of action as first-line treatment for ALK+ NSCLC in a systems biology-based in silico setting. Therapeutic performance mapping system (TPMS) technology was used to characterize the mechanisms of action of brigatinib and alectinib and the impact of potential resistances and drug interferences with concomitant treatments. The analyses indicate that brigatinib and alectinib affect cell growth, apoptosis and immune evasion through ALK inhibition. However, brigatinib seems to achieve a more diverse downstream effect due to a broader cancer-related kinase target spectrum. Brigatinib also shows a robust effect over invasiveness and central nervous system metastasis-related mechanisms, whereas alectinib seems to have a greater impact on the immune evasion mechanism. Based on this in silico head to head study, we conclude that brigatinib shows a predicted efficacy similar to alectinib and could be a good candidate in a first-line setting against ALK+ NSCLC. Future investigation involving clinical studies will be needed to confirm these findings. These in silico systems biology-based models could be applied for exploring other unanswered questions.

Background: Cancer-tumor associated macrophage (TAM)-cancer associated fibroblast (CAF) interactions are an important factor in the tumor microenvironment of hepatocellular carcinoma. Materials and Methods: Hepatic stellate cells (HSCs) were cultured with cancer cell-conditioned medium (Ca.-CM), TAM-CM and CAF-CM, and the expression of CAF markers were evaluated by RT-PCR. Whether HSCs cultured with Ca.-CM, TAM-CM and CAF-CM contributed to the enhanced malignancy of cancer cells was examined using proliferation, invasion and migration assays. Furthermore, the differences between these three types of CM were evaluated using cytokine arrays. Results: HSCs cultured with Ca.-CM, TAM-CM and CAF-CM showed significantly increased mRNA expression of αSMA, FAP and IL-6. All HSCs cultured with each CM exhibited significantly increased proliferation, invasion and migration of cancer cells. The osteopontin concentration was significantly higher in HSCs cultured with TAM-CM than the other CAF-CMs. Osteopontin inhibition significantly reduced osteopontin secretion from HSCs cultured with TAM-CM and suppressed the proliferation and invasion of cancer cells enhanced by HSCs cultured with TAM-CM. Conclusions: We observed enhanced osteopontin secretion from TAMs, and this increased osteopontin further promoted osteopontin secretion from HSCs cultured with TAM-CM, leading to increased malignancy. For the first time, we demonstrated the importance of cancer-TAM-CAF interactions via osteopontin in hepatocellular carcinoma.

Melanoma tumors driven by BRAF mutations often do not respond to BRAF/MEK/ERK pathway inhibitors currently used in treatment. One documented mechanism of resistance is upregulation of SOX2, a transcription factor that is essential for tumor growth and expansion, particularly in melanoma tumors with BRAF mutations. Targeting transcription factors pharmacologically has been elusive for drug developers, limiting treatment options. Here we show that ubiquitin-specific peptidase 9, X-linked (Usp9x), a deubiquitinase (DUB) enzyme controls SOX2 levels in melanoma. Usp9x knockdown in melanoma increased SOX2 ubiquitination, leading to its depletion, and enhanced apoptotic effects of BRAF inhibitor and MEK inhibitors. Primary metastatic melanoma samples demonstrated moderately elevated Usp9x and SOX2 protein expression compared to tumors without metastatic potential. Usp9x knockdown, as well as inhibition with DUB inhibitor, G9, blocked SOX2 expression, suppressed in vitro colony growth, and induced apoptosis of BRAF-mutant melanoma cells. Combined treatment with Usp9x and mutant BRAF inhibitors fully suppressed melanoma growth in vivo. Our data demonstrate a novel mechanism for targeting the transcription factor SOX2, leveraging Usp9x inhibition. Thus, development of DUB inhibitors may add to the limited repertoire of current melanoma treatments.

Pancreatic cancer ranks one of the worst in overall survival outcome with a 5 year survival rate being less than 10%. Pancreatic cancer faces unique challenges in its diagnosis and treatment, such as the lack of clinically validated biomarkers and the immensely immunosuppressive tumor microenvironment. Recently, the LY6 gene family has received increasing attention for its multi-faceted roles in cancer development, stem cell maintenance, immunomodulation, and association with more aggressive and hard-to-treat cancers. A detailed study of mRNA expression of LY6 gene family and its association with overall survival (OS) outcome in pancreatic cancers is lacking. We used publicly available clinical datasets to analyze the mRNA expression of a set of LY6 genes and its effect on OS outcome in the context of the tumor microenvironment and immunomodulation. We used web-based tools Kaplan-Meier Plotter, cBioPortal, Oncomine and R-programming to analyze copy number alterations, mRNA expression and its association with OS outcome in pancreatic cancer. These analyses demonstrated that high expression of LY6 genes is associated with OS and disease free survival (DFS) outcome. High expression of LY6 genes and their association with OS outcome is dependent on the composition of tumor microenvironment. Considering that LY6 proteins are anchored to the outer cell membrane or secreted, making them readily accessible, these findings highlight the potential of LY6 family members in the future of pancreatic cancer diagnosis and treatment.

The stock of therapeutic weapons available in metastatic colorectal cancer (mCRC) has been progressively grown over the years, with improving both survival and patients' clinical outcome: notwithstanding advances in the knowledge of mCRC biology, as well as advances in treatment, fluoropyrimidine antimetabolite drugs have been for 30 years the mainstay of chemotherapy protocols for this malignancy. 5-Fluorouracil (5FU) seems to act differently depending on administration method: elastomer-mediated continuous infusion better inhibits Thymidylate Synthase (TS), an enzyme playing a pivotal role in DNA synthetic pathway. TS overexpression is an acknowledged poor prognosis predicting factor. The simultaneous combination of 5FU and folinate salt synergistically strengthens fluorouracil cytotoxic effect. In our experience, levofolinate and 5FU together in continuous infusion prolong progression free survival of patients suffering from mCRC, moreover decreasing death risk and showing a clear clinical benefit for patients, irrespective of RAS mutational status, primitive tumor side and metastases surgery.

Immunotherapy has been established as a standard of care for patients with malignant melanoma, however, the long-term side-effects of immunotherapy are still emerging. Studies over the last decade have documented increasing reports of endocrine dysfunction following the initiation of immunotherapy. Our study aimed to detect the proportion of men who have low testosterone before, during, and or/after receiving immunotherapy for malignant melanoma, and to determine the proportion of men who receive testosterone replacement therapy after detection of low testosterone. We performed retrospective chart review of patients with malignant melanoma treated with immunotherapy. Low testosterone was identified in 34 out of 49 patients at some point during their treatment with immunotherapy. Despite low testosterone levels in two-thirds of patients, only three patients were treated with testosterone replacement therapy. In addition to laboratory evidence of low testosterone, patients were also symptomatic as 43 out of 49 patients reported fatigue to their providers. Four patients developed hypophysitis and subsequent hypopituitarism, all of whom were receiving Ipilimumab. We conclude that patients with stage 3 or 4 melanoma treated with immunotherapy appear to be at an increased risk of developing testosterone deficiency during their treatment.

Purpose We develop a multi-centric response predictive model using QUS spectral parametric imaging and novel texture-derivate methods for determining tumour responses to neoadjuvant chemotherapy (NAC) prior to therapy initiation. Materials and methods QUS Spectroscopy provided parametric images of mid-band-fit (MBF), spectral-slope (SS), spectral-intercept (SI), average-scatterer-diameter (ASD), and average-acoustic-concentration (AAC) in 78 patients with locally advanced breast cancer (LABC) undergoing NAC. Ultrasound radiofrequency data were collected from Sunnybrook Health Sciences Center (SHSC), University of Texas MD Anderson Cancer Center (MD-ACC), and St. Michaels Hospital (SMH) using two different systems. Texture analysis was used to quantify heterogeneities of QUS parametric images. Further, a second-pass texture analysis was applied to obtain texture-derivate features. QUS, texture- and texture-derivate parameters were determined from both tumour core and a 5-mm tumour margin and were used in comparison to histopathological analysis for developing a response predictive model to classify responders versus non-responders. Model performance was assessed using leave-one-out cross-validation. Three standard classification algorithms including a linear discriminant analysis (LDA), k-nearest-neighbors (KNN), and support vector machines-radial basis function (SVM-RBF) were evaluated. Results A combination of tumour core and margin classification resulted in a peak response prediction performance of 88% sensitivity, 78% specificity, 84% accuracy, 0.86 AUC, 84% PPV, and 83% NPV, achieved using the SVM-RBF classification algorithm. Other parameters and classifiers performed less well running from 66% to 80% accuracy. Conclusions A QUS-based framework and novel texture-derivative method enabled accurate prediction of responses to NAC. Multi-centric response predictive model provides indications of the robustness of the approach to variations due to different ultrasound systems and acquisition parameters.

Objectives: This study investigated harmonized pretreatment volume-based quantitative FDG-PET/CT parameters in breast cancer patients for prognostic value. Results: During a median overall follow-up period of 5.3 years, 91 patients had recurrence and 40 died. Multivariate analysis of ER-positive/HER2-negative patients showed high maximum standardized uptake value (SUVmax) (p = 0.018), high total lesion glycolysis (TLG) (p = 0.010), and clinical N-classification (p = 0.0027) as independent negative predictors of RFS, while high maximum SUVmax (p = 0.037), advanced clinical T-classification (p = 0.030), and advanced TNM stage (p = 0.0067) were independent negative predictors of OS. For recurrence and death in HER2-positive patients, high total TLG (p = 0.037, p = 0.0048, respectively) and advanced TNM stage (p = 0.048, p = 0.046, respectively) were independent prediction factors. In the triple-negative group, independent factors related to recurrence and death were high maximum SUVmax (p = 0.0014, p = 0.0003, respectively) and advanced TNM stage (p < 0.0001, p < 0.0001, respectively). Materials and Methods: Records of 546 stage I–III invasive breast cancer patients, including 344 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative, 110 HER2-positive, and 92 triple-negative cases, treated at four institutions were reviewed retrospectively. Harmonized primary tumor and nodal maximum SUVmax, metabolic tumor volume (MTV), and TLG indicated in pretreatment FDG-PET/CT results were analyzed. Evaluations of relationships of clinicopathological factors, volume-based quantitative parameters, recurrence-free survival (RFS), and overall survival (OS) for each subtype were performed with a Cox proportional hazards model and log-rank test. Conclusions: The results indicated that potential surrogate markers for prognosis in patients with the three main subtypes of operable breast cancer include harmonized pretreatment quantitative volume-based FDG-PET/CT parameters, particularly whole-lesion SUVmax and TLG.

Background: Gastrointestinal stromal tumors (GISTs) quite often co-exist with other primary tumors, as seen in up to 33% of cases. In the literature such occurrences have primarily been described through case reports and rarely through case series, which is not sufficient to prove if there is an association between these two entities. Materials and Methods: We conducted a retrospective study using medical and pathological records from sixty-nine patients who underwent surgical treatment for GIST in a single university surgical department between 2011 and 2019. Seven cases of GIST accompanying a synchronous primary tumor were identified and included in the study. Results: Survival analysis comparing the overall survival of patients with single GIST versus patients with concurrent GIST and another primary tumor, has shown no statistically significant difference between these two groups (p = 0.19). However, when comparing the recurrence rate, patients with synchronous GISTs and another primary tumor have a statistically significant increased possibility for recurrence (p = 0.02). Statistical analysis comparing the size of GISTs between the two groups has shown that patients with single GIST have larger tumors than patients with synchronous tumors (p = 0.048). Conclusions: The synchronous occurrence of GISTs and other intra-abdominal tumors is more common than previously considered, though it is not yet clear if there is a causal association for the concomitant occurrence. Further studies are required to elucidate the genetic and molecular mechanisms of carcinogenesis and progression associating GIST and synchronous tumors.

Genome-wide analysis is widely applied to detect molecular alterations during oncogenesis and tumor progression. We analyzed DNA methylation profiles of hepatocellular carcinoma (HCC), and investigated the clinical role of most heypermethylated of tumor, encodes T-box 15 (TBX15), which was originally involved in mesodermal differentiation. We conducted a genome-wide analysis of DNA methylation of tumor and non-tumor tissue of 15 patients with HCC, and revealed TBX15 was the most hypermethylated gene of tumor (Beta-value in tumor tissue = 0.52 compared with non-tumor tissue). Another validation set, which comprised 58 HCC with radical resection, was analyzed to investigate the relationships between tumor phenotype and TBX15 mRNA expression. TBX15 mRNA levels in tumor tissues were significantly lower compared with those of nontumor tissues (p < 0.0001). When we assigned a cutoff value = 0.5-fold, the overall survival 5-year survival rates of the low-expression group (n = 17) were significantly shorter compared with those of the high-expression group (n = 41) (43.3% vs. 86.2%, p = 0.001). Multivariate analysis identified low TBX15 expression as an independent prognostic factor for overall and disease-free survival. Therefore, genome-wide DNA methylation profiling indicates that hypermethylation and reduced expression of TBX15 in tumor tissue represents a potential biomarker for predicting poor survival of patients with HCC.

Lung cancer is a collection of aggressive tumors generally not diagnosed until late-stage, resulting in high mortality rates. The vast majority of non-small cell lung cancer (NSCLC) patients undergo combinatory chemotherapeutic treatment, which initially reduces tumor growth, but frequently becomes ineffective due to toxicity and resistance. Researchers have identified multiple signaling pathways involved in lung cancer chemoresistance, including cyclooxygenase-2 (COX-2)/microsomal prostaglandin E synthase-1 (mPGES-1) derived prostaglandin E2 (PGE2). While COX-2 inhibitors have shown promise in the clinic, their use is limited due to severe side effects. One novel approach to effectively suppress COX-2 signaling is through microRNA (miRNA). MiRNAs are small-noncoding RNAs commonly misexpressed in cancer. One tumor suppressive miRNA, miR-708-5p, has been shown to repress pro-resistant signaling pathways, including COX-2 and mPGES-1. Here, we demonstrate that chemotherapies reduce COX-2 expression, possibly through induction of miR-708-5p. Moreover, combination treatment of erlotinib (ERL) or paclitaxel (PAC) with miR-708-5p enhances COX-2 and mPGES-1 protein suppression. We also show that combination chemotherapeutic and miR-708-5p treatment intensifies the anti-proliferative and pro-apoptotic effects of ERL and PAC. We also created ERL and PAC resistant lung cancer cell lines, which have increased COX-2 expression and diminished miR-708-5p levels compared to naïve lung cancer cells. While ERL and PAC treatments do not alter resistant cell phenotype alone, combination treatment with miR-708-5p partially restores the chemotherapies’ anti-proliferative effects and fully restores their pro-apoptotic qualities. These data suggest miR-708-5p may have potential combinatory therapeutic value to more efficaciously treat lung tumors while overcoming chemoresistance.

Introduction: Endocrine therapy has played an important role in the management of ER positive breast cancer over recent decades. Despite this, not all patients respond equally to endocrine intervention, which can lead to resistance, associated disease relapse and progression. Previous reports suggest that endocrine agents themselves may induce an invasive phenotype in ER positive breast cancers with low/aberrant expression of E-cadherin. Here we investigate this phenomenon further and provide data supporting a role for the ER co-receptor, PELP-1, in mediating an adverse response to endocrine agents.\ud\udMaterials and Methods: The effects of tamoxifen, fulvestrant and estrogen withdrawal (as a model for aromatase inhibitor therapy) on the invasive and migratory capacity of endocrine-sensitive MCF-7 and T47D cells, in the presence or absence of functional E-cadherin and/or PELP-1 (using siRNA knockdown), was assessed via Matrigel invasion and Boyden chamber migration assays. The effects of these endocrine therapies alongside E-cadherin/PELP-1 modulation on cell proliferation were further assessed by MTT assay. Western blotting using phospho-specific antibodies was performed to investigate signalling pathway changes associated with endocrine-induced changes in invasion and migration.\ud\udResults: Both tamoxifen and fulvestrant induced a pro-invasive and pro-migratory phenotype in ER positive breast cancer cells displaying a high basal expression of PELP-1, which was augmented in the context of poor cell-cell contact. This process occurred in a Src-dependent manner with Src inhibition reversing endocrine induced invasion/migration. While this adverse response was observed using both tamoxifen and fulvestrant therapy, it was not observed under conditions of estrogen withdrawal.\ud\udConclusions: Our data confirms previous reports that anti-estrogens induce an adverse cell phenotype in ER+ breast cancer, particularly in the absence of homotypic cell contact. These results implicate E-cadherin and PELP-1 as potential biomarkers when deciding upon optimum adjuvant endocrine therapy, whereby tumours with high PELP-1/low E-cadherin expression may benefit from estrogen withdrawal therapy via aromatase inhibition, as opposed to ER modulation/antagonism.

Background In epithelial ovarian cancer (EOC), 15-20% of the tumors do not respond to first-line chemotherapy (paclitaxel with platinum-based therapy), and in recurrences this number increases. Our aim is to determine the feasibility of cell proliferation assays of tumor cells isolated from malignant ascites to predict in vitro chemotherapy sensitivity, and to correlate these results with clinical outcome. Materials and methods Ascites was collected from twenty women with advanced EOC. Cell samples were enriched for tumor cells and EOC origin was confirmed by intracellular staining of CK7, surface staining of CA125 and EpCAM, and HE4 gene expression. In vitro sensitivity to chemotherapy was determined in cell proliferation assays using intracellular ATP content as an indirect measure of cell number. In vitro drug response was quantified by calculation of the drug concentration at which cell growth was inhibited with 50%. Clinical outcome was determined using post-treatment CA125 level. Results Cell samples of twenty patients were collected, of which three samples that failed to proliferate were excluded in the analysis (15%). Three other samples were excluded, because clinical outcome could not be determined correctly. In twelve of the fourteen remaining cases (86%) in vitro drug sensitivity and clinical outcome corresponded, while in two samples (14%) there was no correspondence. Conclusions Our study demonstrates the feasibility of drug sensitivity tests using tumor cells isolated from ascites of advanced EOC patients. Larger observational studies are required to confirm the correlation between the in vitro sensitivity and clinical outcome.

Majority of patients with clear-cell renal cell carcinoma (ccRCC) at first line (1L) treatment are classified in the intermediate-risk (IR) subgroup according to International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) score. As these patients have different prognosis, the aim of this study is to better characterize IR patients in order to better tailor the treatment. Retrospective analysis was performed from IGReCC (Institut Gustave Roussy Renal Cell Carcinoma) database. Overall survival (OS) was defined from start of 1L therapy to death or last follow-up. A multivariable Cox model with backward selection procedure (α = 0.01) and a Classification and Regression Tree (CART) analysis were performed to identify which prognostic factors were associated to OS in IR patients. From 2005 to 2017, 777 patients with ccRCC were treated with an anti-VEGF 1L therapy. Among 571 evaluable patients for IMDC score, 290 (51%) were classified as IR. With median follow-up 5.8 years (min: 0, max: 12.4) 212 deaths (73%) were observed and median OS was 25 months. Only platelet count was significantly associated to OS (hazard ratio 1.88 [95% CI 1.27–2.88] p = 0.0017). Median OS for patients with PLT > UNL was 18 months [95% CI 12–23] versus 29 months [95% CI 21.4–35.7] for patients with normal PLT count. The selection of PLT count was confirmed on bootstrap samples and was also selected for the first split of the CART-tree analysis. Patients in the IR group have a heterogeneous prognosis. Elevated PLT count seems identifies a subgroup of patients with poor outcome in the IMDC intermediate-risk population with ccRCC.

Centrosomes amplification is a hallmark of cancer. We hypothesize that 2-methoxyestradiol (2-ME) sensitizes breast cancer (BC) cells to taxanes by targeting amplified centrosomes. We assessed the extent by which 2-ME together with paclitaxel (PTX) induces centrosome alterations with subsequent mitotic catastrophe in different BC subtypes. 2-ME induced a significant reduction in PTX IC50 values in all cells tested ranging from 28–44% (P < 0.05). Treatment with both PTX and 2-ME significantly increased the number of misaligned metaphases compared to PTX alone (34%, 100% and 52% for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). The number of cells with multipolar spindle formation was significantly increased (81%, 220% and 285% for MCF7, MDA-MB231 and SUM 149, respectively; P < 0.05). PTX and 2-ME treatment significantly increased interphase declustering in cancer cells (56% for MCF7, 208% for MDA-MB231 and 218% for SUM149, respectively; P < 0.05) and metaphase declustering (1.4-fold, 1.56-fold and 2.48-fold increase for MCF7, MDA-MB231 and SUM149, respectively; P < 0.05). This report is the first to document centrosome declustering as a mechanism of action of 2-ME and provides a potential approach for reducing taxane toxicity in cancer treated patients.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. For advanced HCC, there is still an unmet need for more effective therapeutic strategies. HCC is typically associated with hypoxia and the hypoxia-inducible factor (HIF) regulatory pathway plays an important role in HCC development and progression. Therefore, we investigated the therapeutic potential of isoform-specific HIF-1α and HIF-2α antisense oligonucleotides (ASOs), along with their effect on the inflammatory and fibrotic component of the tumor microenvironment (TME), in an experimental HCC mouse model. Based on its efficacy and safety, a dosage regimen of 20 mg/kg intraperitoneal injection of HIFα ASO twice per week was selected for further investigation in a preventive and therapeutic setting in a N,N-diethylnitrous amide (DEN)-induced HCC mouse model. DEN administration resulted in 100% tumor formation and HIFα ASO administration led to effective and selective hepatic downregulation of its target genes. HIFα ASO treatment had no effect on tumor numbers, but even enhanced the increased hepatic expression of HCC tumor markers, α-fetoprotein and glypican-3, compared to scrambled control ASO treatment in HCC mice. Especially HIF-1α ASO treatment resulted in an enhanced increase of monocytes and monocyte-derived macrophages in the liver and an enhanced hepatic upregulation of inflammatory markers. Both HIFα ASOs aggravated liver fibrosis in HCC mice compared to scrambled ASO treatment. The observed effects of our dosing regimen for HIF-1α and HIF-2α ASO treatment in the DEN-induced HCC mouse model discourage the use of HIFα isoforms as targets for the treatment of HCC.

Neuroblastoma is the most common extracranial solid tumor in childhood. Patients in high-risk group often have poor outcomes with low survival rates despite several treatment options. This study aimed to identify a genetic signature from gene expression profiles that can serve as prognostic indicators of survival time in patients of high-risk neuroblastoma, and that could be potential therapeutic targets. RNA-seq count data was downloaded from UCSC Xena browser and samples grouped into Short Survival (SS) and Long Survival (LS) groups. Differential gene expression (DGE) analysis, enrichment analyses, regulatory network analysis and machine learning (ML) prediction of survival group were performed. Forty differentially expressed genes (DEGs) were identified including genes involved in molecular function activities essential for tumor proliferation. DEGs used as features for prediction of survival groups included EVX2, NHLH2, PRSS12, POU6F2, HOXD10, MAPK15, RTL1, LGR5, CYP17A1, OR10AB1P, MYH14, LRRTM3, GRIN3A, HS3ST5, CRYAB and NXPH3. An accuracy score of 82% was obtained by the ML classification models. SMIM28 was revealed to possibly have a role in tumor proliferation and aggressiveness. Our results indicate that these DEGs can serve as prognostic indicators of survival in high-risk neuroblastoma patients and will assist clinicians in making better therapeutic and patient management decisions.

The involvement of non-coding RNAs (ncRNAs) in cellular physiology and disease pathogenesis is becoming increasingly relevant in recent years specifically in cancer research. Breast cancer (BC) has become a health concern and accounts for most of the cancer-related incidences and mortalities reported amongst females. In spite of the presence of promising tools for BC therapy, the mortality rate of metastatic BC cases is still high. Therefore, the genomic exploration of the BC subtype and the use of ncRNAs for possible regulation is pivotal. The expression and prognostic values of AURKA gene were assessed by Oncomine, GEPIA, KM-plotter, and bc-GenExMiner v4.4, respectively. Associated proteins and functional enrichment were evaluated by Cytoscape and DAVID databases. Additionally, molecular docking approach was employed to investigate the regulatory role of hsa-miR-32-3p assisted argonaute (AGO) protein of AURKA gene in BC. AURKA gene was highly expressed in patients with BC relative to normal counterpart and significantly correlated with poor survival. The docking result suggested that AURKA could be regulated by hsa-miR-32-3p as confirmed by the reported binding energy and specific interactions. The study gives some insights into role of AURKA and its regulation by microRNAs through AGO protein. It also provides exciting opportunities for cancer therapeutic intervention.

CD44v6, the CD44 isoform mostly involved in cancer cell migration and invasion, has been identified as a functional biomarker and therapeutic target in colon cancer stem cells. We here provide evidence that baseline CD44v6-positive CTC predict treatment failure in patients with metastatic colorectal cancer undergoing first-line chemotherapy. We suggest that CD44v6-positive CTC can be used to early detect intrinsic drug resistance in this cancer type.

Background: Inflammation influences cancer progression by increasing catabolism and impairing nutrient absorption. We compared the prognostic ability of three inflammation-based prognostic scoring systems—the Glasgow prognostic score (GPS), modified GPS (mGPS), and high-sensitivity mGPS (HS-mGPS)—in gastric cancer patients. Materials and Methods: We retrospectively examined 434 curatively resected gastric cancer patients to evaluate the prognostic ability of scoring systems for overall survival (OS) and cancer-specific survival (CSS). Results: OS analysis identified the following independent prognostic factors: GPS model: pathological stage (pStage, p < 0.001), carcinoembryonic antigen (CEA, p = 0.004), and GPS 1 (hazard ratio [HR], 1.929; 95% confidence interval [CI], 1.152-3.228; p = 0.013); mGPS model: body mass index (BMI, p = 0.027), pStage (p < 0.001), and CEA (p < 0.001); HS-mGPS model: BMI (p = 0.029), pStage (p < 0.001), and CEA (p = 0.003). mGPS and HS-mGPS were not independent prognostic factors for OS. CSS analysis of the GPS model identified pStage (p < 0.001), CEA (p = 0.015), and GPS 1 (HR; 2.095, 95% CI; 1.025–4.283; p = 0.043) and 2 (HR, 2.812; 95% CI, 1.111–7.116; p = 0.029) as independent prognostic factors; however, mGPS and HS-mGPS were not independent prognostic factors for CSS. Log-rank tests demonstrated significant differences in OS among patients with GPS 0 vs. 1 (p < 0.001) and 0 vs. 2 (p < 0.001) and in CSS among the three GPS (0 vs. 1; p = 0.005, 0 vs. 2; p < 0.001, 1 vs. 2; p = 0.009). Conclusions: GPS most reliably predicts long-term survival of gastric cancer patients.

AKT, a serine threonine kinase, exists in three different isoforms and is known for regulating several biological processes including tumorigenesis. In this study, we investigated the expression and net effect of the individual isoforms in triple negative breast cancers and response to cisplatin treatment using cellular, mice models and clinical samples. Interestingly, analysis of the expressions of AKT isoforms in clinical samples showed relatively higher expression of AKT1 in primary tissues; whereas lung and liver metastatic samples showed elevated expression of AKT2. Similarly, triple-negative breast cancer cell lines, BT-549 and MDA-MB-231, with high proliferative and invasive properties, displayed higher expression levels of AKT1/2. By modulating AKT isoform expression in MCF-10A and BT-549 cell lines, we found that presence of AKT2 was associated with invasiveness, stemness and sensitivity to drug treatment. It was observed that the silencing of AKT2 suppressed the cancer stem cell populations (CD44high CD24low, ALDH1), mammosphere formation, invasive and migratory potential in MCF-10A and BT-549 cells. It was further demonstrated that loss of function of AKT1 isoform is associated with reduced sensitivity towards cisplatin treatment in triple-negative breast cancers cellular and syngeneic mice models. The decrease in cisplatin treatment response in shAKT1 cells was allied with the upregulation in the expression of transporter protein ABCG2, whereas silencing of ABCG2 restored cisplatin sensitivity in these cells through AKT/SNAIL/ABCG2 axis. In conclusion, our study demonstrated the varied expression of AKT isoforms in triple-negative breast cancers and also confirmed differential role of isoforms in stemness, invasiveness and response towards the cisplatin treatment.

Introduction: Ki-67 expression, a marker of tumor proliferation, is considered a prognostic factor in primary CNS lymphoma (PCNSL). Apparent diffusion coefficient (ADC) parameters have also been proposed as imaging biomarkers for tumor progression and proliferative activity in various malignancies. The aim of this study is to investigate the correlation between ADC parameters, Ki-67 expression, overall survival (OS) and progression free survival (PFS) in PCNSL. Materials and Methods: Patients diagnosed with PCNSL at MD Anderson Cancer Center between Mar 2000 and Jul 2016 and at Ben Taub Hospital between Jan 2012 and Dec 2016 were retrospectively studied. Co-registered ADC maps and post-contrast images underwent whole tumor segmentation. Normalized ADC parameters (nADC) were calculated as the ratio to normal white matter. Percentiles of nADC were calculated and were correlated with Ki-67 using Pearson’s correlation coefficient and clinical outcomes (OS and PFS) using Cox proportional hazards models. Results: Selection criteria yielded 90 patients, 23 patients living with HIV (PLWH) and 67 immunocompetent patients. Above median values for nADCmean, nADC15, nADC75 and nADC95 were associated with improved OS in all patients (p < 0.05). Above median values for nADCmin, nADCmean, nADC1, nADC5 and kurtosis were associated with improved PFS in all patients (p < 0.05). In patients with available Ki-67 expression data (n = 22), nADCmean, nADC15 and nADC75 inversely correlated with Ki-67 expression (p < 0.05). For PLWH, there was no correlation between ADC parameters and Ki-67 expression or clinical outcomes. Conclusions: ADC histogram analysis can predict tumor proliferation and survival in immunocompetent patients with PCNSL, but with limited utility in PLWH.

Conventional cancer chemotherapies are not fully efficacious and do not target tumors, leading to significant treatment-related morbidities. A number of genetically attenuated cancer-targeting bacteria are being developed to safely target tumors in vivo. Here we report the toxicological, tumor-targeting, and efficacy profiles of Salmonella enterica serovar Typhimurium CRC2631 in a syngeneic and autochthonous TRAMP model of aggressive prostate cancer. CRC2631 preferentially colonize primary and metastatic tumors in the TRAMP animals. In addition, longitudinal whole genome sequencing studies of CRC2631 recovered from prostate tumor tissues demonstrate that CRC2631 is genetically stable. Moreover, tumor-targeted CRC2631 generates an anti-tumor immune response. Combination of CRC2631 with checkpoint blockade reduces metastasis burden. Collectively, these findings demonstrate a potential for CRC2631 in cancer immunotherapy strategies.

The canonical Wnt/β-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11-Rad50-Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/β-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/β-catenin signalling through increased β-catenin expression; (3) the functional perturbation of Wnt/β-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3β inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/β-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/β-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of β-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/β-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability.

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

Human breast cancer which affects 1/8 women is rare at a cellular level. Even in the setting of germline BRCA1/BRCA2, which is present in all breast cells, solitary cancers or cancers arising at only several foci occur. The overwhelming majority of breast cells (109–1012 cells) resist transformation. Our hypothesis to explain this rareness of transformation is that mammary oncogenesis is regulated by the cell of origin’s critical window of differentiation so that target cells outside of this window cannot transform. Our novel hypothesis differs from both the multi-hit theory of carcinogenesis and the stem/progenitor cell compartmental theory of tumorigenesis and utilizes two well established murine transgenic models of breast oncogenesis, the FVB/N-Tg (MMTV-PyVT)634Mul/J and the FVB-Tg (MMTV-ErbB2) NK1Mul/J. Tail vein fibroblasts from each of these transgenics were used to generate iPSCs. When select clones were injected into cleared mammary fat pads, but not into non-orthotopic sites of background mice, they exhibited mammary ontogenesis and oncogenesis with the expression of their respective transgenes. iPSC clones, when differentiated along different non-mammary lineages in vitro, were also not able to exhibit either mammary ontogenesis or oncogenesis in vivo. Therefore, in vitro and in vivo regulation of differentiation is an important determinant of breast cancer oncogenesis.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG antibody conjugated via a hydrolysable linker to SN-38, the topoisomerase I-inhibitory active component of irinotecan. We investigated whether Trop-2-expression and homologous recombination repair (HRR) of SN-38-mediated double-strand DNA (dsDNA) breaks play a role in the sensitivity of triple-negative breast cancer (TNBC) to SG. Activation of HRR pathways, as evidenced by Rad51 expression, was assessed in SG-sensitive cell lines with low and moderate Trop-2-expression (SK-MES-1 squamous cell lung carcinoma and HCC1806 TNBC, respectively), compared to a low Trop-2-expressing, less SG-sensitive TNBC cell line (MDA-MB-231). Further, two Trop-2-transfectants of MDA-MB-231, C13 and C39 (4- and 25-fold higher Trop-2, respectively), were treated in mice with SG to determine whether increasing Trop-2 expression improves SG efficacy. SG mediated >2-fold increase in Rad51 in MDA-MB-231 but had no effect in SK-MES-1 or HCC1806, resulting in lower levels of dsDNA breaks in MDA-MB-231. SG and saline produced similar effects in parental MDA-MB-231 tumor-bearing mice (median survival time (MST) = 21d and 19.5d, respectively). However, in mice bearing higher Trop-2-expressing C13 and C39 tumors after Trop-2 transfection, SG provided a significant survival benefit, even compared to irinotecan (MST = 97d vs. 35d for C13, and 81d vs. 28d for C39, respectively; P < 0.0007). These results suggest that SG could provide better clinical benefit than irinotecan in patients with HRR-proficient tumors expressing high levels of Trop-2, as well as to patients with HRR-deficient tumors expressing low/moderate levels of Trop-2.

Ewing sarcoma is an aggressive malignant neoplasm with high propensity for metastasis and poor clinical outcomes. The EWS/Fli1 oncofusion is the disease driver in > 90% of cases, but presents a difficult therapeutic target. Moreover, EWS/Fli1 plays a complex role in disease progression, with inhibitory effects on critical steps of metastasis. Like many other pediatric cancers, Ewing sarcoma is a disease marked by epigenetic dysregulation. Epigenetic mechanisms present alternative targeting opportunities, but their contributions to Ewing sarcoma metastasis and disease progression remain poorly understood. Here, we show that the epigenetic regulators KDM5A and PHF2 promote growth and metastatic properties in Ewing sarcoma, and, strikingly, activate expression many pro-metastatic genes repressed by EWS/Fli1. These genes include L1CAM, which is associated with adverse outcomes in Ewing sarcoma, and promotes migratory and invasive properties. KDM5A and PHF2 retain their growth promoting effects in more metastatically potent EWS/Fli1low cells, and PHF2 promotes both invasion and L1CAM expression in this cell population. Furthermore, KDM5A and PHF2 each contribute to the increased metastatic potency of EWS/Fli1low cells in vivo. Together, these studies identify KDM5A and PHF2 as novel disease-promoting factors, and potential new targets, in Ewing sarcoma, including the more metastatically potent EWS/Fli1low cell population.

Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth.

Cisplatin is a promising therapeutic for the treatment of non-small cell lung cancer (NSCLC). Unfortunately, a significant portion of NSCLC patients relapse due to cisplatin chemoresistance. This chemoresistance is thought to be primarily associated with hypoxia in the tumor microenvironment. Administration of hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) is a promising strategy to alleviate hypoxia in the tumor, which may make cisplatin more effective. In this study, we administered a high O2 affinity, relaxed state (R-state) polymerized hemoglobin (PolyHb) to three different NSCLC cell lines cultured in vitro and implanted in vivo into healthy mice. The R-state PolyHb administered in this study is unable to deliver O2 unless under severe hypoxia which significantly limits its oxygenation potential. In vitro sensitivity studies indicate that the administration of PolyHb increases the effectiveness of cisplatin under hypoxic conditions. Additional animal studies revealed that co-administration of PolyHb with cisplatin attenuated tumor growth without alleviating hypoxia. Analysis of reactive O2 species production in the presence of hypoxic culture indicates that exogenous ROS production by oxidized PolyHb may the mechanism of chemosensitization. This ROS mechanism, coupled with oxygenation, may be a potential chemosensitizing strategy for use in NSCLC treatment.

Background To investigate quantitative ultrasound (QUS) based higher-order texture derivatives in predicting the response to neoadjuvant chemotherapy (NAC) in patients with locally advanced breast cancer (LABC). Materials and methods 100 Patients with LABC were scanned before starting NAC. Five QUS parametric image-types were generated from radio-frequency data over the tumor volume. From each QUS parametric-image, 4 grey level co-occurrence matrix-based texture images were derived (20 QUS-Tex1), which were further processed to create texture derivatives (80 QUS-Tex1-Tex2). Patients were classified into responders and non-responders based on clinical/pathological responses to treatment. Three machine learning algorithms based on linear discriminant (FLD), k-nearest-neighbors (KNN), and support vector machine (SVM) were used for developing radiomic models of response prediction. Results A KNN-model provided the best results with sensitivity, specificity, accuracy, and area under curve (AUC) of 87%, 81%, 82%, and 0.86, respectively. The most helpful features in separating the two response groups were QUS-Tex1-Tex2 features. The 5-year recurrence-free survival (RFS) calculated for KNN predicted responders and non-responders using QUS-Tex1-Tex2 model were comparable to RFS for the actual response groups. Conclusions We report the first study demonstrating QUS texture-derivative methods in predicting NAC responses in LABC, which leads to better results compared to using texture features alone.

The targeted delivery of interleukin-2 to the tumor is gaining attention as an avenue to potentiate the action of T and NK cells at the site of disease. We have previously described the fusion of the L19 antibody, specific to the EDB domain of fibronectin, with human interleukin-2, using a non-covalent homodimeric diabody format. Here, we describe four novel formats for the L19-IL2 fusion, featuring different arrangements of antibody and IL2. A comparative quantitative biodistribution analysis in tumor-bearing mice using radioiodinated proteins revealed that the novel format (L19L19-IL2, with the antibody in single-chain diabody format) exhibited the best biodistribution results. In vitro assays on peripheral blood mononuclear cells showed a decrease activation of regulatory T cells when single IL2 domain was used. In vivo, both L19-IL2 and L19L19-IL2 inhibited tumor growth in immunocompetent mouse models of cancer. T-cell analysis revealed similar levels of CD4+ and FoxP3+ cells, with an expansion of the CD8+ T cell in mice treated with L19-IL2 and L19L19-IL2. The percentage of CD4+ regulatory T cells was markedly decreased with L19L19-IL2 combined with a mouse-specific PD-1 blocker. Collectively, these data indicate that the new L19L19-IL2 format exhibits favorable tumor-homing properties and mediates a potent anti-cancer activity in vivo., Oncotarget, 11 (41), ISSN:1949-2553

Myocardial damage in cancer patients is emphasized as a cause of death; however, there are not many murine cachexia models to evaluate cancer-derived heart disorder. Using the mouse cachexia model that we established previously, we investigated myocardial damage in tumor-bearing mice. In cachexic mice, decreased heart weight and myocardial volume, and dilated left ventricular lumen, and atrophied cardiomyocytes were noted. The cardiomyocytes also showed accumulated 8-hydroxydeoxyguanosine, decreased leucine zipper and EF-hand-containing transmembrane protein-1, and increased microtubule-associated protein light chain3-II. Levels of tumor necrosis factor-α and high-mobility group box-1 proteins in the myocardium were increased, and nuclear factor κB, a signaling molecule associated with these proteins, was activated. When rat cardiomyoblasts (H9c2 cells) were treated with mouse cachexia model ascites and subjected to flux analysis, both oxidative phosphorylation and glycolysis were suppressed, and the cells were in a quiescent state. These results are in good agreement with those previously reported on cancerous myocardial damage. The established mouse cachexia model can therefore be considered useful for analyzing cancer-derived myocardial damage.

The cell-cycle related genes are potential gene targets in understanding the effects of efavirenz (EFV) in lung cancer. The present study aimed at investigating the expression changes of cell-cycle related genes in response to EFV drug treatment in human non-small cell lung carcinoma (A549) and normal lung fibroblast (MRC-5) cells. The loss in nuclear integrity in response to EFV was detected by 4′, 6-diamidino-2-phenylindole (DAPI) staining. Gene expression profiling was performed using human cell cycle PathwayFinder RT2 Profiler™ PCR Array. The expression changes of 84 genes key to the cell cycle pathway in humans following EFV treatment was examined. The R2 PCR Array analysis revealed a change in expression of selected gene targets (including MAD2L2, CASP3, AURKB). This change in gene expression was at least a two-fold between test (EFV treated) and the control. RT-qPCR confirmed the PCR array data. In addition to this, the ATM signaling pathway was shown to be upregulated following EFV treatment in MRC-5 cells. In particular, ATM’s upstream activation resulted in p53 upregulation in normal lung fibroblasts. Interestingly, the p53 signaling pathway was activated irrespective of the repressed ATM pathway in A549 cells as revealed by the Ingenuity Pathway Analysis (IPA). These EFV effects are similar to those of ionizing radiation and this suggests that EFV has anti-tumour properties.

Simon's two-stage design and the admissible two-stage design have been commonly used in practice for single-arm phase II clinical trials when the primary endpoint is binary. The ethical benefit of the two-stage design over the single-stage design is attained by the early termination of the trial when the treatment seems to be inactive. While Simon's optimal design is the two-stage design that minimizes the expected number of subjects under the null hypothesis, the probability of falsely declaring futility after the first stage frequently seems undesirably high. In Simon's minimax design, however, it is often the case that a high proportion of the total planned subjects are evaluated in the first stage, and thus the ethical benefit may not be achieved. In this paper, we propose modified minimax and optimal two-stage designs which guarantee not only type I and II error rates but also reasonable sample size proportions in the first stage, while maintaining the probability of falsely declaring futility under a pre-selected level. The characteristics of the modified two-stage design will be compared with those of Simon's and the admissible two-stage design. The modified minimax design yields a design that requires modest increase in 29% of cases, while the modified optimal design saves 1 to 13 subjects in 81% of cases for β = 0.2. The modified design approach provides investigators with an alternative when the sample sizes of Simon's designs are severely unbalanced or the Type II error is unacceptably high after the first stage.

Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion in vivo following infusion, and represents potential point of intervention to decrease serious toxicities following CAR-T treatment. In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.