Tumor Microenvironment-Responsive Polymeric iRGD and Doxorubicin Conjugates Reduce Spontaneous Lung Metastasis in an Orthotopic Breast Cancer Model.

Tremendous progress has been made in the field of nanomedicine for cancer treatment. However, most of the research to date has been focused on inhibiting primary tumor growth with comparatively less efforts directed towards managing tumor metastasis. Here, we introduce a polymeric conjugate P-DOX-iRGD that not only significantly suppressed primary tumor growth but also substantially inhibited pulmonary metastasis in an orthotopic mouse model of breast cancer. In addition, treatment with P-DOX-iRGD markedly reduced breast cancer-induced splenomegaly and liver hematopoiesis. Interestingly, contrasting results were seen for the free form and polymeric form of DOX in vitro and in vivo, which may be attributed to the enhanced permeability and retention (EPR) effect.

Hot Tub Lung: Case Report and Review of the Literature.

INTRODUCTION: Nontuberculous mycobacteria-related hypersensitivity pneumonitits (NTM-HP), otherwise known as hot tub lung, is an uncommon disease produced by exposure to aerosolized hot tub water containing nontuberculous mycobacteria. Patients usually present with nonspecific, prolonged respiratory symptoms and require a thorough respiratory workup, including radiography and even pulmonary biopsies. CASE PRESENTATION: We present the case of a 58-year-old patient with chronic respiratory symptoms and history of exposure to a hot tub. DISCUSSION: There is little data on why certain patients develop NTM-HP; however, it seems to be an immunologic response to the nontuberculous mycobacteria, not a primary infection. The treatment, as in this case, is typically just hot tub avoidance. CONCLUSION: To our knowledge, this is the first case of NTM-HP reported from Wisconsin. NTM-HP can mimic nontuberculous mycobacterial disease and should be on the differential diagnosis for patients with unclear chronic respiratory problems.

Rare and complicated functional posterior mediastinal paraganglioma.

Functional mediastinal paragangliomas (PGs) are rare, catecholamine-secreting tumours. Surgical resection is the preferred treatment, but it can be complicated by catecholamine-related symptoms, involvement of cardiac structures and/or tumour supply from major blood vessels. We report a case of a man in his 30s with a subcarinal functional PG complicated by all three factors. The PG had arterial supplies from the right coronary and bronchial arteries, with mass effect on the left atrium. Given the high risk of intraoperative bleeding, catecholamine surges and injury to right coronary artery, we attempted a minimally invasive strategy that incorporates best practices from the few published cases on functional PGs. We show that a multidisciplinary approach involving alpha/beta blockade, preoperative embolisation of tumour blood supply, robotic-assisted tumour mobilisation and, if needed, open resection with cardiopulmonary bypass can be a safe strategy in the treatment of functional mediastinal paragangliomas adherent to cardiac structures.

Diffuse interstitial pneumonia-like/macrophage activation syndrome-like changes in patients with COVID-19 correlate with length of illness.

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

Crosstalk between PKCα and PI3K/AKT Signaling Is Tumor Suppressive in the Endometrium.

Protein kinase C (PKC) isozymes are commonly recognized as oncoproteins based on their activation by tumor-promoting phorbol esters. However, accumulating evidence indicates that PKCs can be inhibitory in some cancers, with recent findings propelling a shift in focus to understanding tumor suppressive functions of these enzymes. Here, we report that PKCα acts as a tumor suppressor in PI3K/AKT-driven endometrial cancer. Transcriptional suppression of PKCα is observed in human endometrial tumors in association with aggressive disease and poor prognosis. In murine models, loss of PKCα is rate limiting for endometrial tumor initiation. PKCα tumor suppression involves PP2A-family-dependent inactivation of AKT, which can occur even in the context of genetic hyperactivation of PI3K/AKT signaling by coincident mutations in PTEN, PIK3CA, and/or PIK3R1. Together, our data point to PKCα as a crucial tumor suppressor in the endometrium, with deregulation of a PKCα→PP2A/PP2A-like phosphatase signaling axis contributing to robust AKT activation and enhanced endometrial tumorigenesis.

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer.

MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.

Loss of the Nuclear Pool of Ubiquitin Ligase CHIP/STUB1 in Breast Cancer Unleashes the MZF1-Cathepsin Pro-oncogenic Program.

CHIP/STUB1 ubiquitin ligase is a negative co-chaperone for HSP90/HSC70, and its expression is reduced or lost in several cancers, including breast cancer. Using an extensive and well-annotated breast cancer tissue collection, we identified the loss of nuclear but not cytoplasmic CHIP to predict more aggressive tumorigenesis and shorter patient survival, with loss of CHIP in two thirds of ErbB2+ and triple-negative breast cancers (TNBC) and in one third of ER+ breast cancers. Reduced CHIP expression was seen in breast cancer patient-derived xenograft tumors and in ErbB2+ and TNBC cell lines. Ectopic CHIP expression in ErbB2+ lines suppressed in vitro oncogenic traits and in vivo xenograft tumor growth. An unbiased screen for CHIP-regulated nuclear transcription factors identified many candidates whose DNA-binding activity was up- or downregulated by CHIP. We characterized myeloid zinc finger 1 (MZF1) as a CHIP target, given its recently identified role as a positive regulator of cathepsin B/L (CTSB/L)-mediated tumor cell invasion downstream of ErbB2. We show that CHIP negatively regulates CTSB/L expression in ErbB2+ and other breast cancer cell lines. CTSB inhibition abrogates invasion and matrix degradation in vitro and halts ErbB2+ breast cancer cell line xenograft growth. We conclude that loss of CHIP remodels the cellular transcriptome to unleash critical pro-oncogenic pathways, such as the matrix-degrading enzymes of the cathepsin family, whose components can provide new therapeutic opportunities in breast and other cancers with loss of CHIP expression.Significance: These findings reveal a novel targetable pathway of breast oncogenesis unleashed by the loss of tumor suppressor ubiquitin ligase CHIP/STUB1. Cancer Res; 78(10); 2524-35. ©2018 AACR.

hPaf1/PD2 interacts with OCT3/4 to promote self-renewal of ovarian cancer stem cells.

Cancer stem cells (CSCs), which mediate drug resistance and disease recurrence in several cancers, are therapeutically relevant to ovarian cancer (OC), wherein approximately 80% of patients manifest with tumor recurrence. While there are several markers for ovarian CSCs (OCSCs), the mechanism for their self-renewal maintenance by unique driver/markers is poorly understood. Here, we evaluated the role of hPaf1/PD2, a core component of RNA Polymerase II-Associated Factor (PAF) complex, in self-renewal of OCSCs through marker and functional analyses, including CRISPR/Cas9-silencing of hPaf1/PD2 in OCSCs and provided a possible mechanism for maintenance of OCSCs. Expression of hPaf1/PD2 showed moderate to intense staining in 32.4% of human OC tissues, whereas 67.6% demonstrated basal expression by immunohistochemistry analysis, implying that the minor proportion of cells overexpressing hPaf1/PD2 could be putative OCSCs. Isolated OCSCs showed higher expression of hPaf1/PD2 along with established CSC and self-renewal markers. Knockdown of hPaf1/PD2 in OCSCs resulted in a significant downregulation of CSC and self-renewal markers, and impairment of in vitro tumor sphere (P < 0.05) and colony formation (P = 0.013). Co-immunoprecipitation revealed that OCT3/4 specifically interacts with hPaf1/PD2, and not with other PAF components (Ctr9, Leo1, Parafibromin) in OCSCs, suggesting a complex-independent role for hPaf1/PD2 in OCSC maintenance. Moreover, there was a significant overexpression and co-localization of hPaf1/PD2 with OCT3/4 in OC tissues compared to normal ovary tissues. Our results indicate that hPaf1/PD2 is overexpressed in OCSCs and maintains the self-renewal of OCSCs through its interaction with OCT3/4; thus, hPaf1/PD2 may be a potential therapeutic target to overcome tumor relapse in OC.

Mucins and associated O-glycans based immunoprofile for stratification of colorectal polyps: clinical implication for improved colon surveillance.

Sessile serrated adenoma/polyps (SSA/P) are premalignant lesions of colorectal cancer that are difficult to distinguish histologically from hyperplastic polyps (HP) of minimal to no malignant potential. Specific markers for differentiating SSA/P from HP can aid clinicians for optimizing colon surveillance intervals. The present study investigates the potential of mucins and associated O-glycans to distinguish SSA/P from HP. Expression of colonic mucins (MUC1, MUC4, MUC17, MUC2, and MUC5AC) and O-glycans [Sialyl LewisA (CA19-9) and Tn/Sialyl-Tn on MUC1] were analyzed in HP (n=33), SSA/P (n=39), and tubular adenoma (TA) (n=36) samples by immunohistochemistry. A significantly reduced expression of MUC4 (p=0.0066), elevated expression of MUC17 (p=0.0002), and MUC5AC (p<0.0001) was observed in SSA/P cases in comparison to HP cases. Interestingly, significantly higher number of SSA/P cases (p<0.0001) exhibited MUC5AC expression in the goblet cells as well as filled the crypt lumen compared to only goblet cells in majority of the HP cases. Improved diagnostic potential was revealed by multivariate logistic regression analysis where combinatorial panel of MUC5AC/MUC17 discriminated SSA/P from HP (SN/SP=85/82%). Finally, the decision tree model based marker panel (CA19-9/MUC17/MUC5AC) predicted HP, SSA/P and TA with SN/SP of 58%/95%, 79%/90% and 97%/83%, respectively. Overall, the mucin and associated O-glycan based panel defined in the present study could aid in discriminating SSA/P from HP to devise better colon surveillance strategies.

MUC4 is negatively regulated through the Wnt/ß-catenin pathway via the Notch effector Hath1 in colorectal cancer.

MUC4 is a transmembrane mucin lining the normal colonic epithelium. The aberrant/de novo over-expression of MUC4 is well documented in malignancies of the pancreas, ovary and breast. However, studies have reported the loss of MUC4 expression in the majority of colorectal cancers (CRCs). A MUC4 promoter analysis showed the presence of three putative TCF/LEF sites, implying a possible regulation by the Wnt/ß-catenin pathway, which has been shown to drive CRC progression. Thus, the objective of our study was to determine whether MUC4 is regulated by ß-catenin in CRC. We first knocked down (KD) ß-catenin in three CRC cell lines; LS180, HCT-8 and HCT116, which resulted in increased MUC4 transcript and MUC4 protein. Additionally, the overexpression of stabilized mutant ß-catenin in LS180 and HCT-8 resulted in a decrease in MUC4 expression. Immunohistochemistry (IHC) of mouse colon tissue harboring tubular adenomas and high grade dysplasia showed dramatically reduced Muc4 in lesions relative to adjacent normal tissue, with increased cytosolic/nuclear ß-catenin. Luciferase assays with the complete MUC4 promoter construct p3778 showed increased MUC4 promoter luciferase activity in the absence of ß-catenin (KD). Mutation of all three putative TCF/LEF sites showed that MUC4 promoter luciferase activity was increased relative to the un-mutated promoter. Interestingly, it was observed that MUC4 expressing CRC cell lines also expressed high levels of Hath1, a transcription factor repressed by both active Wnt/ß-catenin and Notch signaling. The KD of ß-catenin and/or treatment with a Notch γ-secretase inhibitor, Dibenzazepine (DBZ) resulted in increased Hath1 and MUC4 in LS180, HCT-8 and HCT116. Furthermore, overexpression of Hath1 in HCT-8 and LS180 caused increased MUC4 transcript and MUC4 protein. Taken together, our results indicate that the Wnt/ß-catenin pathway suppresses the Notch pathway effector Hath1, resulting in reduced MUC4 in CRC.

Loss of N-acetylgalactosaminyltransferase 3 in poorly differentiated pancreatic cancer: augmented aggressiveness and aberrant ErbB family glycosylation.

BACKGROUND: Aberrant glycosylation of several proteins underlie pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. O-glycosylation is initiated by a family of enzymes known as polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts/GALNTs). In this study, we investigated the role of the O-glycosyltransferase GALNT3 in PDAC. METHODS: Immunohistochemistry staining of GALNT3 was performed on normal, inflammatory and neoplastic pancreatic tissues. Several in vitro functional assays such as proliferation, colony formation, migration and tumour-endothelium adhesion assay were conducted in GALNT3 knockdown PDAC cells to investigate its role in disease aggressiveness. Expression of signalling molecules involved in growth and motility was evaluated using western blotting. Effect of GALNT3 knockdown on glycosylation was examined by lectin pull-down assay. RESULTS: N-acetylgalactosaminyl transferase 3 expression is significantly decreased in poorly differentiated PDAC cells and tissues as compared with well/moderately differentiated PDAC. Further, knockdown of GALNT3 resulted in increased expression of poorly differentiated PDAC markers, augmented growth, motility and tumour-endothelium adhesion. Pull-down assay revealed that O-glycans (Tn and T) on EGFR and Her2 were altered in PDAC cells, which was accompanied by their increased phosphorylation. CONCLUSIONS: Our study indicates that loss of GALNT3 occurs in poorly differentiated PDAC, which is associated with the increased aggressiveness and altered glycosylation of ErbB family proteins.

PR55α Subunit of Protein Phosphatase 2A Supports the Tumorigenic and Metastatic Potential of Pancreatic Cancer Cells by Sustaining Hyperactive Oncogenic Signaling.

The protein phosphatase 2 (PP2A) holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Based on loss-of-function analysis using PP2A catalytic inhibitors or inhibition via tumor viral antigens, limited studies suggest that PP2A is a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α (PPP2R2A), a PP2A regulatory subunit, in pancreatic cancer cells compared with normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared with adjacent normal pancreatic tissues (P < 0.0001) and correlated with poor survival of pancreatic cancer patients (P < 0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cell lines resulted in diminished phosphorylation of both AKT and ERK1/2 (MAPK3/1) and decreased protein levels of ß-catenin (CTNNB1). Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice resulted in markedly reduced tumorigenicity (P < 0.001) and distant metastases. Together, these results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK, and Wnt. These studies also provide a basis for exploring PR55α as a diagnostic or therapeutic target in pancreatic cancer. Cancer Res; 76(8); 2243-53. ©2016 AACR.

B7-H1 expression in malignant pleural mesothelioma is associated with sarcomatoid histology and poor prognosis.

INTRODUCTION: B7 homolog 1 (B7-H1; aka programmed cell death 1 ligand 1) is a negative costimulatory molecule that is associated with poor prognosis in many tumor types. Given the poor prognosis and the limited treatments available for mesothelioma, we decided to examine B7-H1 expression and its association with survival in patients with mesothelioma. METHODS: Expression of B7-H1 was determined in 106 patients using a mouse monoclonal antihuman B7-H1 (clone 5H1-A3) antibody with immunohistochemistry. Positive expression was defined as ≥5% positively stained cells. Clinicopathologic features and survival were compared between B7-H1-positive and B7-H1-negative groups. RESULTS: Malignant mesotheliomas of 42 patients (40%) expressed B7-H1. Patients with B7-H1-postive tumors were less likely to be offered or undergo therapeutic surgery (p = 0.03). All sarcomatoid mesotheliomas except one desmoplastic subtype expressed B7-H1. Survival was significantly decreased for patients whose tumors expressed B7-H1 (5 months median, 2-9.5 months interquartile range) compared with those whose tumors did not (14.5 months, 9.25-19 months; p < 0.0001). In a multivariate model, B7-H1 expression and sarcomatoid mesothelioma remained significantly associated with worse survival (risk ratio 1.71, 95% confidence interval 1.03-2.78 [p = 0.04] and risk ratio 2.18, 1.08-4.23 [p = 0.03], respectively). CONCLUSIONS: B7-H1 is expressed in a substantial proportion of malignant pleural mesotheliomas and is associated with poor survival. Almost all malignant pleural mesotheliomas with sarcomatoid differentiation expressed B7-H1. The expression of B7-H1 may have important therapeutic implications for the management of malignant pleural mesothelioma.

Comparison of digital image analysis versus visual assessment to assess survivin expression as an independent predictor of survival for patients with clear cell renal cell carcinoma.

We previously used quantitative digital image analysis to report that high immunohistochemical tumor expression levels of survivin independently predict poor outcome among patients with clear cell renal cell carcinoma. However, given the cumbersome and costly nature of digital image analysis, we evaluated simple visual assessment as an alternative to digital image analysis for assessing survivin as a predictor of clear cell renal cell carcinoma patient outcomes. We identified 310 patients treated surgically for unilateral, sporadic, clear cell renal cell carcinoma at our institution between 1990 and 1994. Survivin expression was quantified independently by digital image analysis and visual assessment in paraffin slides using a commercially available antibody. We examined the agreement between the 2 methods using the kappa statistic and then used Cox regression to compare the ability of the 2 methods to predict renal cell carcinoma death. The kappa statistic comparing high survivin expression determined by digital image analysis versus visual assessment was .68, indicating substantial agreement between the 2 methods. Moreover, even after multivariate adjustment, the association of high survivin expression with risk of renal cell carcinoma death was similar for both visual assessment (risk ratio = 2.01; 95% confidence interval, 1.26-3.22) and digital image analysis (risk ratio = 1.75; 95% confidence interval, 1.10-2.80). Finally, among patients with "moderate risk" (Stage, Size, Grade, and Necrosis scores 3-6) and "high risk" (Stage, Size, Grade, and Necrosis scores 7 or greater) clear cell renal cell carcinoma, high survivin expression determined by visual assessment was significantly associated with poorer survival (P = .006 and P = .017, respectively). Herein, we demonstrate substantial agreement between survivin quantification by digital image analysis and visual assessment. We further confirm that high survivin expression assessed by visual assessment remains an independent predictor of aggressive clear cell renal cell carcinoma behavior. Thus, visual assessment represents an economical, widely available, and reliable method to assess survivin as a predictor of clear cell renal cell carcinoma patient outcomes.