Altered cytoplasmic and nuclear ADP-ribosylation levels analyzed with an improved ADP-ribose binder are a prognostic factor in renal cell carcinoma.

ADP-ribosylation (ADPR) of proteins is catalyzed by ADP-ribosyltransferases, which are targeted by inhibitors (i.e. poly(ADP-ribose) polymerase inhibitors [PARPi]). Although renal cell carcinoma (RCC) cells are sensitive in vitro to PARPi, studies on the association between ADPR levels and somatic loss of function mutations in DNA damage repair genes are currently missing. Here we observed, in two clear cell RCC (ccRCC) patient cohorts (n = 257 and n = 241) stained with an engineered ADP-ribose binding macrodomain (eAf1521), that decreased cytoplasmic ADPR (cyADPR) levels significantly correlated with late tumor stage, high-ISUP (the International Society of Urological Pathology) grade, presence of necrosis, dense lymphocyte infiltration, and worse patient survival (p < 0.01 each). cyADPR proved to be an independent prognostic factor (p = 0.001). Comparably, absence of nuclear ADPR staining in ccRCC correlated with absence of PARP1 staining (p < 0.01) and worse patient outcome (p < 0.05). In papillary RCC the absence of cyADPR was also significantly associated with tumor progression and worse patient outcome (p < 0.05 each). To interrogate whether the ADPR status could be associated with genetic alterations in DNA repair, chromatin remodeling, and histone modulation, we performed DNA sequence analysis and identified a significant association of increased ARID1A mutations in ccRCCcyADPR+++/PARP1+ compared with ccRCCcyADPR-/PARP1- (31% versus 4%; p < 0.05). Collectively, our data suggest the prognostic value of nuclear and cytoplasmic ADPR levels in RCC that might be further influenced by genetic alterations.

Cytoplasmic ADP-ribosylation levels correlate with markers of patient outcome in distinct human cancers.

ADP-ribosylation (ADPR) is a posttranslational modification whose importance in oncology keeps increasing due to frequent use of PARP inhibitors (PARPi) to treat different tumor types. Due to the lack of suitable tools to analyze cellular ADPR levels, ADPR's significance for cancer progression and patient outcome is unclear. In this study, we assessed ADPR levels by immunohistochemistry using a newly developed anti-ADP-ribose (ADPr) antibody, which is able to detect both mono- and poly-ADPR. Tissue microarrays containing brain (n = 103), breast (n = 1108), colon (n = 236), lung (n = 138), ovarian (n = 142), and prostate (n = 328) cancers were used to correlate ADPR staining intensities to clinico-pathological data, including patient overall survival (OS), tumor grade, tumor stage (pT), lymph node status (pN), and the presence of distant metastasis (pM). While nuclear ADPR was detected only in a minority of the samples, cytoplasmic ADPR (cyADPR) staining was observed in most tumor types. Strong cyADPR intensities were significantly associated with better overall survival in invasive ductal breast cancer (p < 0.0001), invasive lobular breast cancer (p < 0.005), and high grade serous ovarian cancer patients (p < 0.01). Furthermore, stronger cytoplasmic ADPR levels significantly correlated with early tumor stage in colorectal and in invasive ductal breast adenocarcinoma (p < 0.0001 and p < 0.01, respectively) and with the absence of regional lymph node metastasis in colorectal adenocarcinoma (p < 0.05). No correlation to cyADPR was found for prostate and lung cancer or brain tumors. In conclusion, our new anti-ADP-ribose antibody revealed heterogeneous ADPR staining patterns with predominant cytoplasmic ADPR staining in most tumor types. Different cyADPR staining patterns could help to better understand variable response rates to PARP inhibitors in the future.

Microfluidic-based immunohistochemistry for breast cancer diagnosis: a comparative clinical study.

Breast cancer is a highly heterogeneous disease. The efficacy of tailored therapeutic strategies relies on the precise detection of diagnostic biomarkers by immunohistochemistry (IHC). Therefore, considering the increasing incidence of breast cancer cases, a concomitantly time-efficient and accurate diagnosis is clinically highly relevant. Microfluidics is a promising innovative technology in the field of tissue diagnostic, enabling for rapid, reliable, and automated immunostaining. We previously reported the microfluidic-based HER2 (human epidermal growth factor receptor 2) detection in breast carcinomas to greatly correlate with the HER2 gene amplification level. Here, we aimed to develop a panel of microfluidic-based IHC protocols for prognostic and therapeutic markers routinely assessed for breast cancer diagnosis, namely HER2, estrogen/progesterone receptor (ER/PR), and Ki67 proliferation factor. The microfluidic IHC protocol for each marker was optimized to reach high staining quality comparable to the standard procedure, while concomitantly shortening the staining time to 16 min-excluding deparaffinization and antigen retrieval step-with a turnaround time reduction up to 7 folds. Comparison of the diagnostic score on 50 formaldehyde-fixed paraffin-embedded breast tumor resections by microfluidic versus standard staining showed high concordance (overall agreement: HER2 94%, ER 95.9%, PR 93.6%, Ki67 93.7%) and strong correlation (ρ coefficient: ER 0.89, PR 0.88, Ki67 0.87; p < 0.0001) for all the analyzed markers. Importantly, HER2 genetic reflex test for all discordant cases confirmed the scores obtained by the microfluidic technique. Overall, the microfluidic-based IHC represents a clinically validated equivalent approach to the standard chromogenic staining for rapid, accurate, and automated breast cancer diagnosis.

Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult.

To explore the general requirement of endothelial mTORC2 during embryonic and adolescent development, we knocked out the essential mTORC2 component Rictor in the mouse endothelium in the embryo, during adolescence and in endothelial cells in vitro. During embryonic development, Rictor knockout resulted in growth retardation and lethality around embryonic day 12. We detected reduced peripheral vascularization and delayed ossification of developing fingers, toes and vertebrae during this confined midgestational period. Rictor knockout did not affect viability, weight gain, and vascular development during further adolescence. However during this period, Rictor knockout prevented skin capillaries to gain larger and heterogeneously sized diameters and remodeling into tortuous vessels in response to FGF2. Rictor knockout strongly reduced extensive FGF2-induced neovascularization and prevented hemorrhage in FGF2-loaded matrigel plugs. Rictor knockout also disabled the formation of capillary-like networks by FGF2-stimulated mouse aortic endothelial cells in vitro. Low RICTOR expression was detected in quiescent, confluent mouse aortic endothelial cells, whereas high doses of FGF2 induced high RICTOR expression that was associated with strong mTORC2-specific protein kinase Cα and AKT phosphorylation. We demonstrate that the endothelial FGF-RICTOR axis is not required during endothelial quiescence, but crucial for midgestational development and sustained and extensive neovascularization in the adult.

Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells.

The activity of a peptide nucleic acid (PNA) targeting cancer-associated microRNA-221 is described. PNAs against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in the cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed both very high affinity for RNA and efficient cellular uptake without the addition of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by PNA resulted in i) lowering of the hybridization levels of miR-221 measured by RT-qPCR, ii) upregulation of p27Kip1 gene expression, measured by RT-qPCR and western blot analysis. The major conclusion of this study is that efficient delivery of anti­miR PNA through a suitable peptide carrier (Rpep­PNA-a221) leads to inhibition of miR-221 activity, altering the expression of miR-221-regulated functions in breast cancer cells.