CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women's Circle of Health Study and the Women's Circle of Health Follow-Up Study.

BACKGROUND: Tumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes. METHODS: We investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women's Circle of Health Study and Women's Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43-12.33). RESULTS: Women with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16-0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12-0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71-1.10). CONCLUSIONS: In contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer. Trial registration Not applicable.

Evaluation of the serotonin pathway as a biomarker in cholangiocarcinoma.

Background: Cholangiocarcinomas (CCAs) are rare and aggressive malignant tumors of the biliary tract. Serotonin (5HT) has tumor-promoting effects in CCA while inhibition of 5HT synthesis can decrease tumor growth. Methods: In this retrospective study, we evaluated the expression of 5HT and tryptophane hydroxylase-1 (TPH-1) in tumor specimens from patients treated with cisplatin plus gemcitabine (CisGem). We included consecutive patients ≥18 years, with locally advanced unresectable, recurrent, or metastatic CCA who were treated with CisGem and had available archival tumor tissue for immunohistochemistry. Formalin-fixed paraffin (FFPE) sections were stained for 5HT and TPH-1. Specimens were evaluated for neuroendocrine features and tumor-infiltrating lymphocytes (TILs). Serum 5HT was measured. Results: We identified 23 patients fulfilling the inclusion criteria. 5HT expression was absent in almost all tumors examined. TPH-1 expression was neither associated with stage or primary tumor location nor predictive of response to CisGem. There was a trend for improved overall survival (OS) in patients whose tumors had high TPH-1 expression. The examined tumor specimens had no neuroendocrine features. Most sections had no TILs. There was a trend for worse OS in patients with high serum 5HT concentration. Conclusions: Tumor TPH-1 expression was not predictive of response to treatment. There was a trend for improved long-term outcomes in patients with high tumor TPH expression and lower serum 5HT concentration.

Photobac derived from bacteriochlorophyll-a shows potential for treating brain tumor in animal models by photodynamic therapy with desired pharmacokinetics and limited toxicity in rats and dogs.

Photobac is a near infrared photosensitizer (PS) derived from naturally occurring bacteriochlorophyll- a, with a potential for treating a variety of cancer types (U87, F98 and C6 tumor cells in vitro). The main objective of the studies presented herein was to evaluate the efficacy, toxicity and pharmacokinetic profile of Photobac in animals (mice, rats and dogs) and submit these results to the United States Food and Drug Administration (US FDA) for its approval to initiate Phase I human clinical trials of glioblastoma, a deadly cancer disease with no long term cure. The photodynamic therapy (PDT) efficacy of Photobac was evaluated in mice subcutaneously implanted with U87 tumors, and in rats bearing C6 tumors implanted in brain. In both tumor types, the Photobac-PDT was quite effective. The long-term cure in rats was monitored by magnetic resonance imaging (MRI) and histopathology analysis. A detailed pharmacology, pharmacokinetics and toxicokinetic study of Photobac was investigated in both non-GLP and GLP facilities at variable doses following the US FDA parameters. Safety Pharmacology studies suggest that there is no phototoxicity, cerebral or retinal toxicity with Photobac. No metabolites of Photobac were observed following incubation in rat, dog, mini-pig and human hepatocytes. Based on current biological data, Photobac-IND received the approval for Phase-I human clinical trials to treat Glioblastoma (brain cancer), which is currently underway at our institute. Photobac has also received an orphan drug status from the US FDA, because of its potential for treating Glioblastoma as no effective treatment is currently available for this deadly disease.

Cadherin-11 increases tumor cell proliferation and metastatic potential via Wnt pathway activation.

During epithelial-mesenchymal transition (EMT) in cancer progression, tumor cells switch cadherin profile from E-cadherin to cadherin-11 (CDH11), which is accompanied by increased invasiveness and metastatic activity. However, the mechanism through which CDH11 may affect tumor growth and metastasis remains elusive. Here, we report that CDH11 was highly expressed in multiple human tumors and was localized on the membrane, in the cytoplasm and, surprisingly, also in the nucleus. Interestingly, ß-catenin remained bound to carboxy-terminal fragments (CTFs) of CDH11, the products of CDH11 cleavage, and co-localized with CTFs in the nucleus in the majority of breast cancer samples. Binding of ß-catenin to CTFs preserved ß-catenin activity, whereas inhibiting CDH11 cleavage led to ß-catenin phosphorylation and diminished Wnt signaling, similar to CDH11 knockout. Our data elucidate a previously unknown role of CDH11, which serves to stabilize ß-catenin in the cytoplasm and facilitates its translocation to the nucleus, resulting in activation of Wnt signaling, with subsequent increased proliferation, migration and invasion potential.

PTEN Loss Expands the Histopathologic Diversity and Lineage Plasticity of Lung Cancers Initiated by Rb1/Trp53 Deletion.

INTRODUCTION: High-grade neuroendocrine tumors of the lung such as SCLC are recalcitrant cancers for which more effective systemic therapies are needed. Despite their histopathologic and molecular heterogeneity, they are generally treated as a single disease entity with similar chemotherapy regimens. Whereas marked clinical responses can be observed, they are short-lived. Inter- and intratumoral heterogeneity is considered a confounding factor in these unsatisfactory clinical outcomes, yet the origin of this heterogeneity and its impact on therapeutic responses is not well understood. METHODS: New genetically engineered mouse models are used to test the effects of PTEN loss on the development of lung tumors initiated by Rb1 and Trp53 tumor suppressor gene deletion. RESULTS: Complete PTEN loss drives more rapid tumor development with a greater diversity of tumor histopathology ranging from adenocarcinoma to SCLC. PTEN loss also drives transcriptional heterogeneity as marked lineage plasticity is observed within histopathologic subtypes. Spatial profiling indicates transcriptional heterogeneity exists both within and among tumor foci with transcriptional patterns correlating with spatial position, implying that the growth environment influences gene expression. CONCLUSIONS: These results identify PTEN loss as a clinically relevant genetic alteration driving the molecular and histopathologic heterogeneity of neuroendocrine lung tumors initiated by Rb1/Trp53 mutations.

Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence.

Cancer cells encounter a hostile tumor microenvironment (TME), and their adaptations to metabolic stresses determine metastatic competence. Here, we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) is induced in hypoxic tumors acquiring metabolic plasticity and invasive phenotype. In mouse models of breast cancer, genetic ablation of PFKFB4 significantly delays distant organ metastasis, reducing local lymph node invasion by suppressing expression of invasive gene signature including integrin ß3. Photoacoustic imaging followed by metabolomics analyses of hypoxic tumors show that PFKFB4 drives metabolic flexibility, enabling rapid detoxification of reactive oxygen species favoring survival under selective pressure. Mechanistically, hypoxic induction triggers nuclear translocation of PFKFB4 accentuating non-canonical transcriptional activation of HIF-1α, and breast cancer patients with increased nuclear PFKFB4 in their tumors are found to be significantly associated with poor prognosis. Our findings imply that PFKFB4 induction is crucial for tumor cell adaptation in the hypoxic TME that determines metastatic competence.

A mitochondrial unfolded protein response inhibitor suppresses prostate cancer growth in mice via HSP60.

Mitochondrial proteostasis, regulated by the mitochondrial unfolded protein response (UPRmt), is crucial for maintenance of cellular functions and survival. Elevated oxidative and proteotoxic stress in mitochondria must be attenuated by the activation of a ubiquitous UPRmt to promote prostate cancer (PCa) growth. Here we show that the 2 key components of the UPRmt, heat shock protein 60 (HSP60, a mitochondrial chaperonin) and caseinolytic protease P (ClpP, a mitochondrial protease), were required for the development of advanced PCa. HSP60 regulated ClpP expression via c-Myc and physically interacted with ClpP to restore mitochondrial functions that promote cancer cell survival. HSP60 maintained the ATP-producing functions of mitochondria, which activated the ß-catenin pathway and led to the upregulation of c-Myc. We identified a UPRmt inhibitor that blocked HSP60's interaction with ClpP and abrogated survival signaling without altering HSP60's chaperonin function. Disruption of HSP60-ClpP interaction with the UPRmt inhibitor triggered metabolic stress and impeded PCa-promoting signaling. Treatment with the UPRmt inhibitor or genetic ablation of Hsp60 inhibited PCa growth and progression. Together, our findings demonstrate that the HSP60-ClpP-mediated UPRmt is essential for prostate tumorigenesis and the HSP60-ClpP interaction represents a therapeutic vulnerability in PCa.

Extracellular sialyltransferase st6gal1 in breast tumor cell growth and invasiveness.

The sialyltransferase ST6GAL1 that adds α2-6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4.

Signal transduction pathways post-translationally regulating nucleotide metabolism remain largely unknown. Guanosine monophosphate reductase (GMPR) is a nucleotide metabolism enzyme that decreases GTP pools by converting GMP to IMP. We observed that phosphorylation of GMPR at Tyr267 is critical for its activity and found that this phosphorylation by ephrin receptor tyrosine kinase EPHA4 decreases GTP pools in cell protrusions and levels of GTP-bound RAC1. EPHs possess oncogenic and tumor-suppressor activities, although the mechanisms underlying switches between these two modes are poorly understood. We demonstrated that GMPR plays a key role in EPHA4-mediated RAC1 suppression. This supersedes GMPR-independent activation of RAC1 by EPHA4, resulting in a negative overall effect on melanoma cell invasion and tumorigenicity. Accordingly, EPHA4 levels increase during melanoma progression and inversely correlate with GMPR levels in individual melanoma tumors. Therefore, phosphorylation of GMPR at Tyr267 is a metabolic signal transduction switch controlling GTP biosynthesis and transformed phenotypes.

A phase I study of the anaplastic lymphoma kinase inhibitor ceritinib in combination with gemcitabine-based chemotherapy in patients with advanced solid tumors.

In this phase I, dose-escalation study, we sought to determine the maximum tolerated dose (MTD) of the anaplastic lymphoma kinase/c-ROS oncogene 1 receptor (ALK/ROS1) inhibitor ceritinib in combination with gemcitabine-based chemotherapy in patients with advanced solid tumors. Secondary objectives were characterization of the safety profile, pharmacokinetics and preliminary efficacy of these combinations, and identification of potential biomarkers of efficacy. Ceritinib was combined with gemcitabine (Arm 1), gemcitabine/nab-paclitaxel (Arm 2) or gemcitabine/cisplatin (Arm 3). Drug concentrations in plasma were measured by tandem mass spectrometric detection (LC-MS/MS). We analyzed archival tumor tissue for ALK, ROS1, hepatocyte growth factor receptor (c-MET) and c-Jun N-terminal kinase (JNK) expression by immunohistochemistry. Arm 2 closed early secondary to toxicity. Twenty-one patients were evaluable for dose-limiting toxicity (DLT). There was one DLT in Arm 1 (grade 3 ALT increase) and three DLTs in Arm 3 (grade 3 acute renal failure, grade 3 thrombocytopenia, grade 3 dyspnea). The MTD of ceritinib was determined to be 600 mg (Arm 1) and 450 mg orally daily (Arm 3). Main toxicities were hematologic, constitutional and gastrointestinal as expected by the chemotherapy backbone. The apparent clearance for ceritinib decreased substantially after repeated dosing; cisplatin did not significantly affect the pharmacokinetics of ceritinib. The overall response rate was 20%; the median progression-free survival was 4.8 months. Three out of five response-evaluable cholangiocarcinoma patients had clinical benefit. Increased expression of c-MET was associated with a lack of clinical benefit. Ceritinib in combination with gemcitabine and gemcitabine/cisplatin has a manageable toxicity profile. Further development of this strategy in tumors with ALK or ROS1 fusions is warranted.

Body fatness and breast cancer risk in relation to phosphorylated mTOR expression in a sample of predominately Black women.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway promoted by positive energy imbalance and insulin-like growth factors can be a mechanism by which obesity influences breast cancer risk. We evaluated the associations of body fatness with the risk of breast cancer varied with phosphorylated (p)-mTOR protein expression, an indication of the pathway activation. METHODS: Women with newly diagnosed breast cancer (n = 715; 574 [80%] Black and 141 [20%] White) and non-cancer controls (n = 1983; 1280 [64%] Black and 713 [36%] White) were selected from the Women's Circle of Health Study. Surgical tumor samples among the cases were immunostained for p-mTOR (Ser2448) and classified as p-mTOR-overexpressed, if the expression level ≥ 75th percentile, or p-mTOR-negative/low otherwise. Anthropometrics were measured by trained staff, and body composition was determined by bioelectrical impedance analysis. Odds ratios (ORs) of p-mTOR-overexpressed tumors and p-mTOR-negative/low tumors compared to controls were estimated using polytomous logistic regression. The differences in the associations by the p-mTOR expression status were assessed by tests for heterogeneity. RESULTS: Cases with p-mTOR-overexpressed tumors, but not cases with p-mTOR-negative/low tumors, compared to controls were more likely to have higher body mass index (BMI), percent body fat, and fat mass index (P-heterogeneity < 0.05), although the OR estimates were not significant. For the measurement of central adiposity, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 2.52, 95% CI = 1.46 to 4.34) and Q4 (OR = 1.99, 95% CI = 1.12 to 3.50) of waist circumference (WC) compared to controls. Similarly, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 1.82, 95% CI = 1.11 to 2.98) and Q4 (OR = 1.81, 95% CI = 1.11 to 2.98) of WHR compared to controls. These associations of WC and waist-to-hip ratio (WHR) did not differ by tumor p-mTOR status (P-heterogeneity = 0.27 and 0.48, respectively). CONCLUSIONS: Our findings suggest that in this population composed of predominately Black women, body fatness is associated with breast cancer differently for p-mTOR overexpression and p-mTOR negative/low expression. Whether mTOR plays a role in the obesity and breast cancer association warrants confirmation by prospective studies.

GEN-1 in Combination with Neoadjuvant Chemotherapy for Patients with Advanced Epithelial Ovarian Cancer: A Phase I Dose-escalation Study.

PURPOSE: GEN-1 (phIL-12-005/PPC), an IL12 plasmid formulated with polyethyleneglycol-polyethyleneimine cholesterol lipopolymer, has preclinical activity when combined with platinum-taxane intravenous chemotherapy and administered intraperitoneally in epithelial ovarian cancer (EOC) models. OVATION I was a multicenter, nonrandomized, open-label phase IB trial to evaluate the safety, preliminary antitumor activity, and immunologic response to GEN-1 in combination with neoadjuvant chemotherapy (NACT) carboplatin-paclitaxel in patients with advanced EOC. PATIENTS AND METHODS: A total of 18 patients with newly diagnosed stage IIIC and IV EOC were enrolled. A standard 3+3 dose-escalation design tested four GEN-1 doses (36, 47, 61, 79 mg/m2) to determine the maximum tolerated dose and dose-limiting toxicities (DLTs). GEN-1 was administered in eight weekly intraperitoneal infusions starting at cycle 1 week 2 in combination with three 21-day cycles of NACT carboplatin AUC 6 and weekly paclitaxel 80 mg/m2. RESULTS: The most common treatment-emergent adverse events at least possibly related were nausea, fatigue, abdominal pain/cramping, anorexia, diarrhea, and vomiting. Eight patients experience grade 4 neutropenia attributed to NACT. No DLTs occurred. A total of 14 patients were evaluable for response and 12 (85.7%) had radiological response (two complete response and 10 partial response) prior to debulking; nine were R0 at debulking and one patient had complete pathologic response. IL12 and its downstream cytokine, IFNγ, increased in peritoneal washings but not as much in blood. Increased levels of myeloid dendritic cells and T-effector memory cells in peritoneal fluid, plus elevated CD8+ T cells and reduced immunosuppression within the tumor microenvironment were found. A median time to treatment failure of 18.4 months (95% confidence interval, 9.2-24.5) was observed in the intention-to-treat population. CONCLUSIONS: Adding GEN-1 to standard NACT is safe, appears active, and has an impact on the tumor microenvironment.

Porfimer Sodium Versus PS785 for Photodynamic Therapy (PDT) of Lung Cancer Xenografts in Mice.

BACKGROUND: Lung cancer is the greatest cause of cancer mortality in the United States, necessitating ongoing improvements in current treatment techniques. Photodynamic therapy (PDT) involves the interaction between a photosensitizer, light, and oxygen. The resulting release of reactive oxygen species causes tumor necrosis. It has been used as an endoscopic technique for the palliation of lung cancer. Porfimer sodium (Photofrin) is the only Food and Drug Administration-approved photosensitizer for PDT but has limited depth of penetration and produces prolonged skin phototoxicity. Multiple newer photosensitizers are in development, including PS785. The effectiveness of PS785 was compared with porfimer sodium in the treatment of human lung cancer xenografts in mice. METHODS: Human non-small cell lung cancer (NSCLC) xenografts were established in severe combined immunodeficient mice and grouped into small (3-5 mm) and large tumors (6-10 mm). PS785 or porfimer sodium was administered intravenously, and PDT was executed at 24, 48, or 72 h after injection. The primary endpoint was the delay of tumor regrowth after PDT. RESULTS: Porfimer sodium and PS785 produced statistically similar delays of tumor regrowth after PDT when small tumors were treated at 24 and 48 h. At 72 h, PS785 performed better in small tumors. However, for large tumors, PS785 produced no delay in tumor regrowth at any time point. CONCLUSIONS: PS785 and porfimer sodium were able to effectively treat NSCLC to a depth of ≤5 mm. However, porfimer sodium was more effective in treating NSCLC tumors to a depth of 6-10 mm. Further efforts are required to produce photosensitizers that will facilitate PDT of larger tumors.

High intratumoral CD8+ T-cell infiltration is associated with improved survival in prostate cancer patients undergoing radical prostatectomy.

BACKGROUND: A high density of CD8+ tumor infiltrating lymphocytes (TILs) is associated with improved survival in multiple cancers, but its prognostic role in prostate cancer remains controversial. The aim of our study was to evaluate the prognostic value of CD8+ TILs in prostate cancer patients undergoing radical prostatectomy (RP). We hypothesized that elevated density of CD8+ TILs in the RP specimen would correlate with improved clinical outcomes. This information may be helpful for future immunotherapy clinical trial design and treatment selection. METHODS: Tumor microarrays constructed from 230 patients with localized prostate cancers who underwent RP from 2006 to 2012 at Roswell Park Comprehensive Cancer Center were analyzed retrospectively using immunohistochemistry. CD8+ cell density was evaluated using a computerized scoring system. The cohorts were separated by CD8+ TIL density at the 25th percentile (i.e., low 7 or pT3/4). The median follow-up time was 8.4 years. High CD8+ TIL density was associated with improved 5-year overall survival (98% vs. 91%, p = .01) and prostate cancer-specific survival (99% vs. 95%, p = .04) compared with patients with low CD8+ TIL density. There was a trend toward higher 5-year biochemical recurrence-free survival and metastasis-free survival in the cohort of patients with high CD8+ TIL density (52% vs. 38% and 86% vs. 73%, respectively), although the difference did not reach statistical significance (p = .18 and p = .05, respectively). In a multivariate analysis high CD8+ TIL density was an independent favorable prognostic factor for overall survival (hazards ratio = 0.38; 95% confidence interval: 0.17-0.87; p = .02). In contrast to the prognostic value of CD8+ TIL density, the CD8+ cell density in the matched normal prostate tissue was not associated with any clinical outcomes. CONCLUSION: Intratumoral CD8+ T-cell infiltration in the RP specimen is independently associated with improved survival after RP in this high-risk prostate cancer cohort. Pre-RP immunomodulation that promotes intratumoral CD8+ cytotoxic T-cell infiltration may be beneficial for this population.

Sonodynamic therapy in combination with photodynamic therapy shows enhanced long-term cure of brain tumor.

This article presents the construction of a multimodality platform that can be used for efficient destruction of brain tumor by a combination of photodynamic and sonodynamic therapy. For in vivo studies, U87 patient-derived xenograft tumors were implanted subcutaneously in SCID mice. For the first time, it has been shown that the cell-death mechanism by both treatment modalities follows two different pathways. For example, exposing the U87 cells after 24 h incubation with HPPH [3-(1'-hexyloxy)ethyl-3-devinyl-pyropheophorbide-a) by ultrasound participate in an electron-transfer process with the surrounding biological substrates to form radicals and radical ions (Type I reaction); whereas in photodynamic therapy, the tumor destruction is mainly caused by highly reactive singlet oxygen (Type II reaction). The combination of photodynamic therapy and sonodynamic therapy both in vitro and in vivo have shown an improved cell kill/tumor response, that could be attributed to an additive and/or synergetic effect(s). Our results also indicate that the delivery of the HPPH to tumors can further be enhanced by using cationic polyacrylamide nanoparticles as a delivery vehicle. Exposing the nano-formulation with ultrasound also triggered the release of photosensitizer. The combination of photodynamic therapy and sonodynamic therapy strongly affects tumor vasculature as determined by dynamic contrast enhanced imaging using HSA-Gd(III)DTPA.

USP1 Regulates TAZ Protein Stability Through Ubiquitin Modifications in Breast Cancer.

The Hippo signaling pathway is an evolutionarily conserved pathway that was initially discovered in Drosophila melanogaster and was later found to have mammalian orthologues. The key effector proteins in this pathway, YAP/TAZ, are often dysregulated in cancer, leading to a high degree of cell proliferation, migration, metastasis and cancer stem cell populations. Due to these malignant phenotypes it is important to understand the regulation of YAP/TAZ at the protein level. Using an siRNA library screen of deubiquitinating enzymes (DUBs), we identified ubiquitin specific peptidase 1 (USP1) as a novel TAZ (WWTR1) regulator. We demonstrated that USP1 interacts with TAZ and increases TAZ protein stability. Conversely, loss of function of USP1 reduces TAZ protein levels through increased poly-ubiquitination, causing a decrease in cell proliferation and migration of breast cancer cells. Moreover, we showed a strong positive correlation between USP1 and TAZ in breast cancer patients. Our findings facilitate the attainment of better understanding of the crosstalk between these pathways and may lead to potential therapeutic interventions for breast cancer patients.

Body fatness and mTOR pathway activation of breast cancer in the Women's Circle of Health Study.

Energy imbalance has an important role in breast cancer prognosis. Hyperactive mechanistic Target of Rapamycin (mTOR) pathway is associated with breast tumor growth, but the extent to which body fatness is associated with mTOR pathway activities in breast cancer is unclear. We performed immunostaining for mTOR, phosphorylated (p)-mTOR, p-AKT, and p-p70S6K in tumor tissue from 590 women (464 African Americans/Blacks and 126 Whites) with newly diagnosed invasive breast cancer in the Women's Circle of Health Study. Anthropometric measures were taken by study staff, and body composition was measured by bioelectrical impedance analysis. Linear regressions were used to estimate percent differences in protein expression between categories of body mass index (BMI), waist circumference, waist/hip ratio, fat mass, fat mass index, and percent body fat. We observed that BMI ≥ 35.0 vs. <25 kg/m2 was associated with 108.3% (95% CI = 16.9%-270.9%) and 101.8% (95% CI = 17.0%-248.8%) higher expression in p-mTOR and normalized p-mTOR, i.e., p-mTOR/mTOR, respectively. Quartiles 4 vs. 1 of waist/hip ratio was associated with 41.8% (95% CI = 5.81%-89.9%) higher mTOR expression. Similar associations were observed for the body fat measurements, particularly in patients with estrogen receptor-negative (ER-) tumors, but not in those with ER+ tumors, although the differences in associations were not significant. This tumor-based study found positive associations between body fatness and mTOR pathway activation, evident by a p-mTOR expression, in breast cancer. Our findings suggest that mTOR inhibition can be a treatment strategy to prevent the recurrence of these tumors in obese individuals.

Targeting EphA2 in Bladder Cancer Using a Novel Antibody-Directed Nanotherapeutic.

Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.

Proliferative potential and response to nivolumab in clear cell renal cell carcinoma patients.

Background: Biomarkers predicting immunotherapy response in metastatic renal cell cancer (mRCC) are lacking. PD-L1 immunohistochemistry is a complementary diagnostic for immune checkpoint inhibitors (ICIs) in mRCC, but has shown minimal clinical utility and is not used in routine clinical practice. Methods: Tumor specimens from 56 patients with mRCC who received nivolumab were evaluated for PD-L1, cell proliferation (targeted RNA-seq), and outcome. Results: For 56 patients treated with nivolumab as a standard of care, there were 2 complete responses and 8 partial responses for a response rate of 17.9%. Dividing cell proliferation into tertiles, derived from the mean expression of 10 proliferation-associated genes in a reference set of tumors, poorly proliferative tumors (62.5%) were more common than moderately (30.4%) or highly proliferative (8.9%) counterparts. Moderately proliferative tumors were enriched for PD-L1 positive (41.2%), compared to poorly proliferative counterparts (11.4%). Objective response for moderately proliferative (29.4%) tumors was higher than that of poorly (11.4%) proliferative counterparts, but not statistically significant (p = .11). When cell proliferation and negative PD-L1 tumor proportion scores were combined statistically significant results were achieved (p = .048), showing that patients with poorly proliferative and PD-L1 negative tumors have a very low response rate (6.5%) compared to moderately proliferative PD-L1 negative tumors (30%). Conclusions: Cell proliferation has value in predicting response to nivolumab in clear cell mRCC patients, especially when combined with PD-L1 expression. Further studies which include the addition of progression-free survival (PFS) along with sufficiently powered subgroups are required to further support these findings.

Clonality and antigen-specific responses shape the prognostic effects of tumor-infiltrating T cells in ovarian cancer.

CD8+ tumor-infiltrating lymphocytes (TILs) are not all specific for tumor antigens, but can include bystander TILs that are specific for cancer-irrelevant epitopes, and it is unknown whether the T-cell repertoire affects prognosis. To delineate the complexity of anti-tumor T-cell responses, we utilized a computational method for de novo assembly of sequences from CDR3 regions of 369 high-grade serous ovarian cancers from TCGA, and then applied deep TCR-sequencing for analyses of paired tumor and peripheral blood specimens from an independent cohort of 99 ovarian cancer patients. Strongly monoclonal T-cell repertoires were associated with favorable prognosis (PFS, HR = 0.65, 0.50-0.84, p = 0.003; OS, HR = 0.61, 0.44-0.83, p = 0.006) in TCGA cohort. In the validation cohort, we discovered that patients with low T-cell infiltration but low diversity or focused repertoires had clinical outcomes almost indistinguishable from highly-infiltrated tumors (median 21.0 months versus 15.9 months, log-rank p = 0.945). We also found that the degree of divergence of the peripheral repertoire from the TIL repertoire, and the presence of detectable spontaneous anti-tumor immune responses are important determinants of clinical outcome. We conclude that the prognostic significance of TILs in ovarian cancer is dictated by T-cell clonality, degree of overlap with peripheral repertoire, and the presence of detectable spontaneous anti-tumor immune response in the patients. These immunological phenotypes defined by the TCR repertoire may provide useful insights for identifying "TIL-low" ovarian cancer patients that may respond to immunotherapy.