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Background: miRNA therapeutics have gained attention during the past decade. These oligonucleotide treatments can modulate the expression of miRNAs in vivo and could be used to correct the imbalance of gene expression found in human diseases such as obesity, metabolic syndrome, and atherosclerosis. The in vivo efficacy of current anti-miRNA technologies hindered by physiological and cellular barriers to delivery into targeted cells and the nature of miRNAs that allows one to target an entire pathway that may lead to deleterious off-target effects. For these reasons, novel targeted delivery systems to inhibit miRNAs in specific tissues will be important for developing effective therapeutic strategies for numerous diseases including atherosclerosis. Methods: We used pH low-insertion peptide (pHLIP) constructs as vehicles to deliver microRNA-33-5p (miR-33) antisense oligonucleotides to atherosclerotic plaques. Immunohistochemistry and histology analysis was performed to assess the efficacy of miR-33 silencing in atherosclerotic lesions. We also assessed how miR-33 inhibition affects gene expression in monocytes/macrophages by single-cell RNA transcriptomics. Results: The anti-miR-33 conjugated pHLIP constructs are preferentially delivered to atherosclerotic plaque macrophages. The inhibition of miR-33 using pHLIP-directed macrophage targeting improves atherosclerosis regression by increasing collagen content and decreased lipid accumulation within vascular lesions. Single-cell RNA sequencing analysis revealed higher expression of fibrotic genes ( Col2a1, Col3a1, Col1a2, Fn1 , etc) and tissue inhibitor of metalloproteinase 3 ( Timp3 ) and downregulation of Mmp12 in macrophages from atherosclerotic lesions targeted by pHLIP-anti-miR-33. Conclusions: This study provides proof of principle for the application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33 in macrophages that avoid the deleterious effects in other metabolic tissues. This may open new therapeutic opportunities for atherosclerosis-associated cardiovascular diseases via selective delivery of other protective miRNAs.

Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity.The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed.We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

Acidity is a useful biomarker for the targeting of metabolically active cells in tumors. pH Low Insertion Peptides (pHLIPs) sense the pH at the surfaces of tumor cells and can facilitate intracellular delivery of cell-permeable and cell-impermeable cargo molecules. In this study we have shown the targeting of malignant lesions in human bladders by fluorescent pHLIP agents, intracellular delivery of amanitin toxin by pHLIP for the inhibition of urothelial cancer cell proliferation, and enhanced potency of pHLIP-amanitin for cancer cells with 17p loss, a mutation frequently present in urothelial cancers. Twenty-eight ex-vivo bladder specimens, from patients undergoing robotic assisted laparoscopic radical cystectomy for bladder cancer, were treated via intravesical incubation for 15-60 minutes with pHLIP conjugated to indocyanine green (ICG) or IR-800 near infrared fluorescent (NIRF) dyes at concentrations of 4-8 μM. White light cystoscopy identified 47/58 (81%) and NIRF pHLIP cystoscopy identified 57/58 (98.3%) of malignant lesions of different subtypes and stages selected for histopathological processing. pHLIP NIRF imaging improved diagnosis by 17.3% (p < 0.05). All carcinoma-in-situ cases missed by white light cystoscopy were targeted by pHLIP agents and were diagnosed by NIRF imaging. We also investigated the interactions of pHLIP-amanitin with urothelial cancer cells of different grades. pHLIP-amanitin produced concentration- and pH-dependent inhibition of the proliferation of urothelial cancer cells treated for 2 hrs at concentrations up to 4 μM. A 3-4x enhanced cytotoxicity of pHLIP-amanitin was observed for cells with a 17p loss after 2 hrs of treatment at pH6. Potentially, pHLIP technology may improve the management of urothelial cancers, including imaging of malignant lesions using pHLIP-ICG for diagnosis and surgery, and the use of pHLIP-amanitin for treatment of superficial bladder cancers via intravesical instillation.

The acidic pH of tumors profoundly inhibits effector functions of activated CD8 + T-cells. We hypothesize that this is a physiological process in immune regulation, and that it occurs within lymph nodes (LNs), which are likely acidic because of low convective flow and high glucose metabolism. Here we show by in vivo fluorescence and MR imaging, that LN paracortical zones are profoundly acidic. These acidic niches are absent in athymic Nu/Nu and lymphodepleted mice, implicating T-cells in the acidifying process. T-cell glycolysis is inhibited at the low pH observed in LNs. We show that this is due to acid inhibition of monocarboxylate transporters (MCTs), resulting in a negative feedback on glycolytic rate. Importantly, we demonstrate that this acid pH does not hinder initial activation of naïve T-cells by dendritic cells. Thus, we describe an acidic niche within the immune system, and demonstrate its physiological role in regulating T-cell activation., T-cell activation primarily occurs in the lymph nodes, highly organized and specialized secondary lymphoid organs. Here the authors show that the acidic extracellular pH in lymph node paracortical zones limits cytokine production by effector T-cells, but does not alter their activation by antigen-presenting cells.

Aims In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid–base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. Methods and results Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl−/HCO3− exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3− and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. Conclusions Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.

Hypoxia and tissue acidification occur in the macrophage-rich regions of advanced atherosclerotic lesions due to the higher oxygen demand of activated immune cells and insufficient oxygen supply. Our group and others originally identified microRNA-33 (miR-33) as critical regulator of cellular lipid homeostasis and lipoprotein metabolism controlling the development of atherosclerosis. Our prior work has demonstrated that pH Low-Insertion Peptides (pHLIP) can be used to direct miR-33 inhibitors to acidic microenvironments and protect against kidney fibrosis. Here we utilize anti-miR-33 conjugated pHLIP constructs to target macrophages located in atherosclerosis plaques. The inhibition of miR-33 using pHLIP-directed targeting increased collagen content and decreased lesional lipid accumulation within atherosclerotic plaques in a murine model of atherosclerosis regression. Single cell RNA sequencing analysis revealed higher expression of fibrotic genes (Col2a1, Col3a1, Fn1, Dcn, etc) and tissue inhibitor of metalloproteinase 3 (Timp3), and downregulation of matrix metallopeptidase 12 (Mmp12) in macrophages from atherosclerotic lesions targeted by pHLIP- anti-miR-33. These results suggest a potential application of pHLIP for treating advanced atherosclerosis via pharmacological inhibition of miR-33.

The advantages of targeted therapy have motivated many efforts to find distinguishing features between the molecular cell surface landscapes of diseased and normal cells. Typically, the features have been proteins, lipids or carbohydrates, but other approaches are emerging. In this discussion, we examine the use of cell surface acidity as a feature that can be exploited by using pH-sensitive peptide folding to target agents to diseased cell surfaces or cytoplasms.

Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can partially prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in 2 mouse models of kidney disease. These effects were not related to changes in circulating leukocytes because bone marrow transplants from miR-33–deficient animals did not have a similar impact on disease progression. Most important, targeted delivery of miR-33 peptide nucleic acid inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease, This work was supported by grants from the NIH (R35HL135820 to CFH, R01HL105945 and R01HL135012 to YS, GM073857 to OAA and YKR, and P30 DK079310 to the George M. O’Brien Kidney Center at Yale), the American Heart Association (16EIA27550005 to CFH, 16GRNT26420047 to YS, and 17SDG33110002 to NR), the Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research MicroRNA-based Therapeutic Strategies in Vascular Disease (to CFH), and the University of Connecticut START PPOC award (to RB) Ministerio de Economia y Competitividad (MINECO) (SAF2015-66107-R to SL), Carlos III Health Institute (ISCIII) European Regional Development Fund (PI17/01513 to DRP); cofunded by the European Regional Development Fund; and supported by Instituto de Salud Carlos III REDinREN RD12/0021/0009 and RD16/0009/0016 (to SL and DRP), Comunidad de Madrid “NOVELREN” B2017/BMD3751 (to SL and DRP), a grant-in-aid from the Spanish Society of Nephrology (Fundacion Senefro 2017 to SL), and Fundacion Renal “Inigo Alvarez de Toledo” (to SL), all from Spain. The Centro de Biologia Molecular “Severo Ochoa” (CBMSO) receives institutional support from Fundacion “Ramon Areces.” SL was supported by the “Salvador de Madariaga” grant PRX15/00119 from the Spanish Ministry of Educacion, Cultura y Deporte

OBJECTIVE: To improve visualization of upper tract urothelial carcinomas. Previous studies using the novel pH low insertion peptide (pHLIP) variant 3 (Var3) conjugated to indocyanine green (ICG) have demonstrated high sensitivity and specificity for imaging of bladder urothelial carcinoma. Here, we describe a novel approach for the imaging of upper tract urothelial carcinomas using ICG-Var3 pHLIP. METHODS: Twelve ex-vivo upper urinary tract specimens were irrigated with ICG-Var 3 pHLIP for 15 minutes and then examined using a white light laparoscopic camera followed by near infrared fluorescent (NIRF) imaging using a Stryker 1588 AIM imaging system. Standard histopathologic evaluation was performed and findings were correlated with white light and ICG-Var3 NIRF imaging. One patient who underwent radical nephrectomy for renal cell carcinoma was used as a negative control. RESULTS: Nineteen lesions were identified on histopathologic evaluation in 10 patients, including 82% high-grade urothelial carcinoma and 18% low-grade urothelial carcinoma. Nineteen (100%) malignant lesions were identified using NIRF imaging, while 15 (78.9%) lesions were identified using conventional white light examination. The sensitivity of ICG-Var3 pHLIP NIRF imaging was 100% compared to 78.9% white light examination. Both modalities are 100% specific. Benign collecting systems and ureters did not show uptake of the pHLIP construct. CONCLUSION: In this feasibility study, the ICG-Var3 pHLIP imaging agent demonstrated superior diagnostic performance compared to conventional white light examination. While additional studies are required for validation and in-vivo translation, pHLIP-based imaging represents a promising tool to improve the evaluation and management of upper tract urothelial carcinoma. UROLOGY 139: 134–140, 2020.

The pH (low) insertion peptides (pHLIPs) target acidity at the surfaces of cancer cells and show utility in a wide range of applications, including tumor imaging and intracellular delivery of therapeutic agents. Here we report pHLIP constructs that significantly improve the targeted delivery of agents into tumor cells. The investigated constructs include pHLIP bundles (conjugates consisting of two or four pHLIP peptides linked by polyethylene glycol) and Var3 pHLIPs containing either the nonstandard amino acid, γ-carboxyglutamic acid, or a glycine−leucine−leucine motif. The performance of the constructs in vitro and in vivo was compared with previous pHLIP variants. A wide range of experiments was performed on nine constructs including (i) biophysical measurements using steady-state and kinetic fluorescence, circular dichroism, and oriented circular dichroism to study the pH-dependent insertion of pHLIP variants across the membrane lipid bilayer; (ii) cell viability assays to gauge the pH-dependent potency of peptide-toxin constructs by assessing the intracellular delivery of the polar, cell-impermeable cargo molecule amanitin at physiological and low pH (pH 7.4 and 6.0, respectively); and (iii) tumor targeting and biodistribution measurements using fluorophore-peptide conjugates in a breast cancer mouse model. The main principles of the design of pHLIP variants for a range of medical applications are discussed.

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-Fluorescence Insertion REporter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation in vivo. Imaging and targeted cargo delivery should each have clinical applications.

Poly (ADPribose) polymerase inhibitors (PARPi) have shown great promise in the treatment of cancer, however, their current clinical use has been largely limited to homologous recombination-deficient (HRD) tumors. While these drugs are efficacious as monotherapies, durable responses beyond 12 months are uncommon and their activity against HRD-negative tumors is limited. These findings have prompted great interest in combining PARPi’s with chemotherapy to increase the duration of response in HRD-positive tumors and expand the activity of these drugs against HRD-negative tumors which comprise a much greater fraction of the total number of cancer cases each year. While such combinations are highly active against tumors independent of HRD status, they are also extremely cytotoxic against bone marrow cells. As such, dose reductions ranging from 5- to 20-fold are required when PARPi’s are combined with chemotherapy which has resulted in favorable safety profiles but limited efficacy. Tumor-targeting strategies could overcome this efficacy barrier but the technologies developed thus far are limited by numerous issues, including: (a) lack of universal, non-saturating tumor-targeting mechanisms which limit their use to specific tumor types and their corresponding antigens, (b) inability to deliver therapeutically relevant levels of drug(s) directly into tumor cells, and (c) insufficient tumor penetration leading to sub-optimal tumor exposure. Herein, we report the development of alphalex, a novel tumor-targeted drug delivery platform which allows the efficient and selective delivery of PARPi’s into tumors with significant normal-tissue sparing. Tumor-targeting is achieved using a pH- sensitive peptide which forms an alpha-helix in cellular membrane under low pH conditions associated with the tumor microenvironment. Under these conditions, the peptide translocates across cancer cell membranes to deliver the PARPi directly into the cytoplasm. We demonstrate that alphalex peptide-based conjugates can be combined safely and effectively with both cytotoxic chemotherapies and radiation therapy (RT) and that this approach can be used to selectively kill both HRD-positive and –negative tumors with significant sparing of the bone marrow. The safety and efficacy of the approach was demonstrated in both engineered and patient-derived xenografts (PDXs) in vivo using two FDA-approved PARPi’s across two independent laboratories. These data highlight an entirely new approach to apply PARPi’s against solid tumors independent of HRD status. Furthermore, our approach can be applied to a diverse range of DNA repair inhibitors for potent and selective chemo/radio-sensitization in a tissue-agnostic manner. Citation Format: Vishwas Paralkar, Robert J. Aiello, Dan Marshall, Johanna Csengery, Patricia Bourassa, Qing Zhang, Brett S. Robinson, Lori Lopresti-Morrow, Jane Bechtold, Laurie Tylaska, Timothy Paradis, Gregory Slaybaugh, Hanna Visca, Anna Moshnikova, Dhammika Weerakkody, Oleg Andreev, Yana K. Reshetnyak, Donald Engelman, Ranjit Bindra, Peter Glazer, Per Hellsund. Targeting solid tumor acidic microenvironment with an alphalex PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2981.

Introduction: The detection of urothelial carcinomas of the bladder and upper tract is not satisfactory. Incomplete findings by contemporary imaging modalities provide a challenge in patient care, so there is a definitive need to improve diagnosis and treatment methods for urothelial carcinomas. Experimental procedures: ICG (indocyanine green) near infrared fluorescent (NIRF) dyes were conjugated with a pH-Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells. In a few cases IR800 pHLIP® was used. Urinary neoplasm (kidney, ureter and bladder) specimens obtained through radical surgery were irrigated and incubated ex-vivo with ICG pHLIP® followed by fluorescence imaging. Pathology findings were correlated with the ICG pHLIP® NIRF imaging. Results: In the upper urothelial tract, nine of ten patients with nephroureterectomy had urothelial carcinomas (UTUC). Eighteen (100%) malignant lesions were found in NIRF imaging. Only thirteen (72%) of malignant lesions were found under white light macroscopic examination. ICG pHLIP® NIRF imaging gave a 28% increase in diagnosis of UTUC in ex-vivo specimens. There was no non-specific uptake in the non-tumoral tissue. The ICG pHLIP® imaging agent identified malignant upper tract urothelial lesions with 100% specificity and sensitivity. In the lower urinary tract, thirty-eight radical cystectomy specimens were incubated with pHLIP® conjugated with one of the NIRF dyes (ICG or IR800). In the thirty-eight bladders, eighty-one lesions were identified using fluorescent pHLIP®(specificity (97%) and sensitivity (100%)). White light cystoscopy did not detect 20.5% of lesions identified under NIRF cystoscopy (p Conclusions: The ICG pHLIP® NIRF revealed additional lesions not seen with white light ex-vivo cystoscopy or preoperative ureteroscopy. pHLIP® based agents promise to improve diagnostic accuracy for urothelial carcinomas and may enable targeted treatment. Citation Format: Borivoj Golijanin, Anna Moshnikova, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak, Ali Amin, Dragan Golijanin. Ex-vivo targeting of urothelial carcinomas by fluorescent pHLIP imaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1399.

Introduction: Fluorescence imaging has applications in medicine for many image-guided procedures such as mapping and visualization of lymph nodes, targeting and marking cancerous lesions and assessment of tumor margins. Several fluorescent molecules are approved for clinical use including the ICG (indocyanine green) near infrared (NIR) fluorescent dye. We conjugated ICG to a pH Low Insertion Peptide (pHLIP®), which senses and targets the acidity at the surfaces of cancer cells, and evaluated ICG pHLIP® as a targeted imaging agent for tumor visualization. Our construct is progressing toward clinical trials. Experimental Procedures: ICG pHLIP® was manufactured for proof-of-concept tumor-targeting imaging studies in mice, and toxicity evaluation in mice, dogs and rats. Tumor targeting was assessed in various human and murine tumor models. Biodistribution was evaluated at different time points from 5 min to 48 hours after a single intravenous administration of 0.5 mg/kg of ICG pHLIP®. The distribution of the agent within tumors was evaluated in tumor sections. Results: The fluorescence of ICG is enhanced about 15 times when pHLIP® tethers it to a cell membrane, significantly improving the contrast index. No toxicity is seen at 25-30x of the proposed human dose, evaluated in mice and dogs. ICG pHLIP® demonstrates excellent tumor targeting, including targeting of 1-3 mm sized tumors in breast. About 10-15% ID/g was observed within tumors at 4 hours p.i., and the signal persists for up to 24 hours with a slight decline at 48 hours. The background signal in major organs decayed after agent administration. The highest uptake, of about 50% ID/g, was observed in liver at 4 hrs p.i. and decayed after that. An excellent correlation was established between ICG pHLIP® NIRF imaging and H&E histopathology at the indicated tumor location. Conclusions: ICG pHLIP® is a promising new imaging agent for fluorescence-guided surgical applications. It is on the path to clinical translation for accurate resection of breast tumors. Since pHLIP® targeting of tumors is based on the general principle of cancer cell surface acidity, ICG pHLIP® is an agent that may be used in a range of fluorescence-guided surgical interventions. Citation Format: Troy Crawford, Anna Moshnikova, Sean Roles, Lukas M. Carter, Jason S. Lewis, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak. ICG pHLIP: A novel agent for fluorescence-guided surgery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1956.

Cancer is a complex disease with a range of genetic and biochemical markers within and among tumors, but a general tumor characteristic is extracellular acidity, which is associated with tumor growth and development. Acidosis could be a universal marker for cancer imaging and the delivery of therapeutic molecules, but its promise as a cancer biomarker has not been fully realized in the clinic. We have discovered a unique approach for the targeting of acidic tissue using the pH-sensitive folding and transmembrane insertion of pH (low) insertion peptide (pHLIP). The essence of the molecular mechanism has been elucidated, but the principles of design need to be understood for optimal clinical applications. Here, we report on a library of 16 rationally designed pHLIP variants. We show how the tuning of the biophysical properties of peptide–lipid bilayer interactions alters tumor targeting, distribution in organs, and blood clearance. Lead compounds for PET/single photon emission computed tomography and fluorescence imaging/MRI were identified, and targeting specificity was shown by use of noninserting variants. Finally, we present our current understanding of the main principles of pHLIP design.

Bladder cancer is the fifth most common in incidence and one of the most expensive cancers to treat. Early detection greatly improves the chances of survival and bladder preservation. The pH low insertion peptide (pHLIP) conjugated with a near-infrared fluorescent dye [indocyanine green (ICG)] targets low extracellular pH, allowing visualization of malignant lesions in human bladder carcinoma ex vivo. Cystectomy specimens obtained after radical surgery were immediately irrigated with nonbuffered saline and instilled with a solution of the ICG pHLIP construct, incubated, and rinsed. Bladders were subsequently opened and imaged, the fluorescent spots were marked, and a standard pathological analysis was carried out to establish the correlation between ICG pHLIP imaging and white light pathological assessment. Accurate targeting of bladder lesions was achieved with a sensitivity of 97%. Specificity is 100%, but reduced to 80% if targeting of necrotic tissue from previous transurethral resections or chemotherapy are considered as false positives. The ICG pHLIP imaging agent marked high-grade urothelial carcinomas, both muscle invasive and nonmuscle invasive. Carcinoma in situ was accurately diagnosed in 11 cases, whereas only four cases were seen using white light, so imaging with the ICG pHLIP peptide offers improved early diagnosis of bladder cancers and may also enable new treatment alternatives.

Acidification of extracellular space promotes tumor development, progression, and invasiveness. pH (low) insertion peptides (pHLIP® peptides) belong to the class of pH-sensitive membrane peptides, which target acidic tumors and deliver imaging and/or therapeutic agents to cancer cells within tumors. Ex vivo fluorescent imaging of tissue and organs collected at various time points after administration of different pHLIP® variants conjugated with fluorescent dyes of various polarity was performed. Methods of multivariate statistical analyses were employed to establish classification between fluorescently labeled pHLIP® variants in multidimensional space of spectral parameters. The fluorescently labeled pHLIP® variants were classified based on their biodistribution profile and ability of targeting of primary tumors. Also, submillimeter-sized metastatic lesions in lungs were identified by ex vivo imaging after intravenous administration of fluorescent pHLIP® peptide. Different cargo molecules conjugated with pHLIP® peptides can alter biodistribution and tumor targeting. The obtained knowledge is essential for the design of novel pHLIP®-based diagnostic and therapeutic agents targeting primary tumors and metastatic lesions.

A series of cyclic peptides containing a number of tryptophan (W) and glutamic acid (E) residues were synthesized and evaluated as pH-sensitive agents for targeting of acidic tissue and pH-dependent cytoplasmic delivery of molecules. Biophysical studies revealed the molecular mechanism of peptides action and localization within the lipid bilayer of the membrane at high and low pHs. The symmetric, c[(WE)4WC] and asymmetric, c[E4W5C], cyclic peptides translocated amanitin, a polar cargo molecule of similar size, across the lipid bilayer and induced cell death in a pH- and concentration-dependent manner. Fluorescently-labelled peptides were evaluated for targeting of acidic 4T1 mammary tumors in mice. The highest tumor to muscle ratio (5.6) was established for asymmetric cyclic peptide, c[E4W5C], at 24 hours after intravenous administration. pH-insensitive cyclic peptide c[R4W5C], where glutamic acid residues (E) were replaced by positively charged arginine residues (R), did not exhibit tumor targeting. We have introduced a novel class of cyclic peptides, which can be utilized as a new pH-sensitive tool in investigation or targeting of acidic tissue.

We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7-6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1-6.4, whereas in nonmetastatic tumors, it was 6.7-6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible.

Progress in nanomedicine depends on the development of nanomaterials and targeted delivery methods. In this work, we describe a method for the preferential targeting of gold nanoparticles to a tumor in a mouse model. The method is based on the use of the pH Low Insertion Peptide (pHLIP), which targets various imaging agents to acidic tumors. We compare tumor targeting by nonfunctionalized nanogold particles with nanogold–pHLIP conjugates, where nanogold is covalently attached to the N terminus of pHLIP. Our most important finding is that both intratumoral and i.v. administration demonstrated a significant enhancement of tumor uptake of gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-insensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles. The pHLIP technology can substantially improve the delivery of gold nanoparticles to tumors by providing specificity of targeting, enhancing local concentration in tumors, and distributing nanoparticles throughout the entire tumor mass where they remain for an extended period (several days), which is beneficial for radiation oncology and imaging.

NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.

Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using (31)P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications.

Malignant tumors exhibit elevated uptake and use of glucose, as well as metabolite buildup and hypoxia due to inadequate blood supply, leading to tumor acidosis. The extracellular acidity is linked to tumor progression. The most effective pH-sensitive tumor targeting agents should sense pH at the surface of cancer cells, where it is expected to be the lowest. pHLIPs® (pH (Low) Insertion Peptides) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors in BALB/c mice with three Alexa546 labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs.

Currently, chemical bifunctional cross-linkers are regarded as promising therapeutic agents capable of affecting cell metabolism. Depending on the nature of the active groups and on the length of their mediating spacer, these cross-linkers have been shown to influence mitochondrial functions, the cell cycle and cell death. The current study was aimed to assay cellular effects of a cross-linker with 'zero'-length spacer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). When added to cultures of transformed cells, EDC induced a G2/M blockade followed by cell death. Analysis of the molecular targets revealed that alteration of the cell cycle was caused by EDC-induced interchain cross-linking within double-stranded DNA. Administration of EDC to animals with experimental tumors increased their life span. The analysis of tumor cells from EDC-treated mice showed up-regulation of p21/WAF1, disturbance of tumor cell cytokinesis and, hence, cell death. Thus, both in vitro and in vivo, EDC exhibits cytotoxic activity, which may be of potential therapeutic use.

Soluble Fas antigen can protect cells against Fas-mediated apoptosis. High level soluble Fas antigen characteristic for blood of patients with autoimmune disease or cancer is believed to prevent the elimination of autoimmune lymphocytes or tumor cells. Here we first report that human recombinant FasDeltaTM, i.e. soluble Fas generated by alternative splicing of the intact exon 6, is capable of inducing death of transformed cells by "reverse" apoptotic signaling via transmembrane Fas ligand. FasDeltaTM, as well as transmembrane Fas antigen, can be either monomeric or oligomeric, and both its forms are efficient in blocking Fas-mediated apoptosis, although the cytotoxic activity is exhibited solely by the latter. An in vivo analysis of soluble Fas antigen showed that unlike in healthy controls, nearly the total FasDeltaTM present in sera of rheumatoid arthritis patients was oligomeric. This resulted in suppression of cell proliferation in the experimental sera and in its promotion in controls. Thus, oligomerization/depolymerization of soluble Fas antigen can regulate its activity and contribute to the pathogenesis of autoimmune diseases and cancer.

Studies indicating an important role of the TNF-receptor family in control of cell proliferation, differentiation, and death have drastically increased in number in recent years. The main function of many members of this family is cell death triggering, and this is apparently the only function for some of them. Studies on the molecular mechanisms of cell death activated by members of the TNF-receptor family revealed and identified proteins directly or indirectly associated with TNF receptors. Pathways of cytotoxic signal transduction by some members of the TNF-Rs family based on currently proven protein-protein interactions and the role of distinct proteins in these processes are summarized in this review.

Introduction: The pH (Low) Insertion Peptides (pHLIP® peptides) show utility in a wide variety of medical applications due to their ability to target cancer cells by exploiting the acidity that is ubiquitous to tumor tissue. Targeting tumor acidity offers many advantages over traditional biomarker targeting and has allowed pHLIP® peptides to target primary tumor tissue as well as sub-millimeter metastases. Previously, pHLIP®s conjugated to PET/SPECT tracers or fluorescent labels have found applications in diagnostic nuclear imaging and fluorescence-guided surgery. The purpose of this study was to evaluate a diverse collection of pHLIP® variants, including those incorporating non-standard amino acids, consisting of two or four pHLIP®s linked together, and one of de novo design, in order to determine the pHLIP® peptide best suited for the targeting and intracellular delivery of therapeutic cargo molecules to primary tumors and metastases. Methods: Biophysical experiments, namely fluorescence and circular dichroism spectroscopy, using liposomes as model membranes, were used to study the localization of pHLIP® variants at the bilayer surface at high pH, the tertiary structure and orientation within the membrane of the folded variants at low pH, the parameters of pH-dependent peptide insertion, and the rate at which insertion occurs. In vitro experiments, wherein the polar toxin amanitin was conjugated to the membrane-inserting end of the pHLIP® variants via a cleavable disulfide bond, were used to study intracellular cargo delivery and the resulting inhibition of cancer cell proliferation. In vivo experiments, wherein the fluorescent dye Alexa Fluor 546 was conjugated to the non-inserting end of the pHLIP® variants via a non-cleavable bond, were used to study biodistribution and tumor uptake. Results: All investigated pHLIP® variants demonstrated the behavior characteristic of wild-type pHLIP® with some differences in membrane affinity and membrane insertion profiles, namely pKa of insertion and cooperativity. Inhibition of cancer cell proliferation was induced by the pH–dependent translocation of amanitin across the plasma membrane by the pHLIP® variants. Biophysical measurements, values of EC50 obtained at pH 6.0 and pH 7.4, and fluorescence imaging of triple-negative breast tumors in mice using pHLIP® peptides were analyzed collectively. Conclusions: Different pHLIP® variants are best suited for different purposes, such as the intracellular delivery of highly or moderately toxic polar or hydrophobic payloads to primary tumor tissue as well as metastases. pHLIP® Variant 3 (Var3), exhibited the most favorable properties for the targeted delivery of amanitin to tumor tissue. Acknowledgement: This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM073857 to OAA and YKR. Citation Format: Linden C. Wyatt, Anna Moshnikova, Troy Crawford, Oleg A. Andreev, Yana K. Reshetnyak. Utility of pH (low) insertion peptide (pHLIP® peptide) variants in drug delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5137. doi:10.1158/1538-7445.AM2017-5137

Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA, accounting for ~40,000 deaths annually. The dismal prognosis for PDAC is largely due to its late diagnosis. Currently, the most sensitive diagnosis of PDAC requires invasive procedures, such as endoscopic ultrasonography, which has inherent risks and accuracy that is highly operator dependent. Here we took advantage of a general characteristic of solid tumors, the acidic microenvironment that is generated as a by-product of metabolism, to develop a novel approach of using pH (Low) Insertion Peptides (pHLIPs) for imaging of PDAC. We show that fluorescently labeled pHLIPs can localize and specifically detect PDAC in human xenografts as well as PDAC and PanIN lesions in genetically engineered mouse models. This novel approach may improve detection, differential diagnosis and staging of PDAC.

Physical association of proteins that underlies cytotoxic signal induction and transduction suggests a possibility of regulating cell response by modifying protein-protein interactions. For protein complexing, chemical cross-linking agents have been traditionally used. However, the ability of various cross-linkers to induce and modify cell responses, cell death in particular, is still obscure. We have undertaken the investigation to test the apoptosis-inducing and modifying properties of the homobifunctional cross-linkers-dimethyl suberimidate (DMS) and 1,5-bis(succinimido-oxycarbonyloxy)pentane (BSOCOP). The functional groups of these cross-linkers are different but both are able to interact with available amino groups. It was shown that bifunctional cross-linkers, unlike their monofunctional analogues, are capable of inducing cell death in transformed cells, thus indicating the crucial role of cross-linking in cell killing. DMS- and BSOCOP-treated cells were shown to undergo cell death by apoptosis, though the signaling pathways were distinct. DMS inhibited bcl-X(L) and bak but not bax gene expression, while BSOCOP potentiated bax mRNA synthesis immediately after application. Cell pre-incubation with DMS, but not with BSOCOP, resulted in an increasing sensitivity to TNF, although activities of anti-Fas cytotoxic antibodies were then inhibited. Thus, this study has demonstrated for the first time that chemical cross-linkers are capable of inducing apoptosis by themselves and modifying the TNF-dependent and Fas-mediated cell death that may have potential therapeutic significance.

Introduction: Extracellular acidosis promotes tumor development, progression and invasiveness. A combination of effects acidifies tumor cell interiors, so to maintain intracellular pH, cells pump out lactic acid and protons, acidifying the extracellular space. In addition, overexpression of carbonic anhydrases on the surfaces of cancer cells further contributes to an acidification of the environment especially near the cell surface. Thus, the pH near tumor cell surfaces is expected to be the lowest, and it increases with distance from the membrane. pH (Low) Insertion Peptides (pHLIP® peptides) pertain to the class of pH-sensitive agents able of sensing pH at the cellular surface and delivery of imaging and therapeutic agents to the cancer cells in tumors. Experimental Procedures: We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, SNARF, conjugated to the pH Low Insertion Peptide (pHLIP® peptide). The measurement tool was validated using cancer cells grown in spheroids, in mice and in excised tumors. Also, we performed imaging of primary tumors and metastatic lesions testing various fluorescent pHLIP® constructs on animal tumor models and human tissue. Results: pHLIP® is a platform technology using pH-triggered, membrane-associated folding for targeting acidic tumors. In contrast to other approaches developed to measure extracellular pH and target acidic environments (mostly in endosomes, where the pH is around 5.0-5.5), the pHLIP® technology has the capability to “sense” and target pH at the surfaces of tumor cells. We established that the pH at the surface of cancer cells is 0.2-0.4 pH units lower (in some parts of tumor it reaches values of pH6.0-6.1) than bulk extracellular pH measured up to now and demonstrated that the surface pH is sensitive to cell glycolytic activity. The approach was sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by injection of glucose. In response to low pH at the surface of cancer cells, pHLIP® peptides insert across the cell membrane and undergo coil-helix transition, which gives a high cooperativity for the transition compared with any pH-dependent diffusion-limited process, and allow targeting imaging agents to cancer cells in tumors. Even small differences in pH between normal and cancerous cells can be distinguished, and since both the pK of insertion and the cooperativity can be tuned, the system can be refined to target specific pH ranges. We have demonstrated targeting of acidic tumors in mice tumor models and in human tissue. Conclusions: Even individual cancer cell maintain acidity on its surface and could be detected in a well perfused areas. pHLIP® technology can be used in vivo and ex vivo on tissue specimens and opens both a path to medical utility and an exploration of other functions of acidity on cell surfaces. The leading fluorescent pHLIP® construct was selected for translation to clinics for diagnostic and surgical resection of primary tumors and submillimeter metastatic lesions. Citation Format: Anna Moshnikova, Michael Anderson, Ramona-Cosmina Adochite, Donald M. Engelman, Oleg A. Andreev, Yana K. Reshetnyak. pHLIP® technology for imaging acidic tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4250.

The pH (low) insertion peptide (pHLIP) family enables targeting of cells in tissues with low extracellular pH. Here, we show that ischemic myocardium is targeted, potentially opening a new route to diagnosis and therapy. The experiments were performed using two murine ischemia models: regional ischemia induced by coronary artery occlusion and global low-flow ischemia in isolated hearts. In both models, pH-sensitive pHLIPs [wild type (WT) and Var7] or WT-pHLIP–coated liposomes bind ischemic but not normal regions of myocardium, whereas pH-insensitive, kVar7, and liposomes coated with PEG showed no preference. pHLIP did not influence either the mechanical or the electrical activity of ischemic myocardium. In contrast to other known targeting strategies, the pHLIP-based binding does not require severe myocardial damage. Thus, pHLIP could be used for delivery of pharmaceutical agents or imaging probes to the myocardial regions undergoing brief restrictions of blood supply that do not induce irreversible changes in myocytes.

pHLIP-FIRE (Fluorescence Insertion REporter) is a novel diagnostic tool based on pHLIP (pH Low Insertion Peptide), a water soluble membrane peptide that interacts with membranes by forming a transmembrane helix at low pH. The fluorescence of pHLIP-FIRE is activated by separation of mutually quenching or donor-quencher fluorescent groups via the cleavage of a disulfide-linker when the C-terminus moves into the highly reducing environment inside the cell. Targeting studies in mice bearing breast tumors showed strong targeting of pHLIP-FIRE in vivo, giving a contrast index of 20 with respect to background. This contrast is a significant enhancement in comparison with traditional fluorescent pHLIP constructs. Experiments in vitro and in cultured cells show that the time-course of dequenching in pHLIP-FIRE is pH-dependent and quite slow ( ∼ 1 - 2 days). Direct imaging with pHLIP-FIRE gives the first demonstration of pHLIP insertion and cargo translocation in vivo. Our technology shows promise as a tool for cancer diagnosis or image-guided surgery, and the demonstration of cargo delivery into tumors may lead to a new generation of targeted chemotherapeutic delivery.

Fas/FasL system is involved in pathogenesis of a variety of autoimmune diseases. In overwhelming majority of situations alterations in Fas and FasL expression are viewed in frames of Fas-mediated apoptosis. In the present work we tested a possible involvement of Fas-ligand-mediated "reverse signaling" in pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We show that high level of sFas in RA patient blood correlates with a high activity of disease; in SLE patients with elevated sFas level there was a correlation between sFas concentration and leucopenia, and tissue and organ damage. We showed for the first time that at high concentrations in serum sFas is present in oligomeric form. Oligomeric sFas demonstrated cytotoxicity in lymphocyte primary culture and in transformed cells, while non-toxic recombinant Fas-ligand partially blocked this effect. Besides, immunohistochemical analysis of PBLs and injured synovia of RA patients revealed the high expression of Fas-ligand. All this together allow assuming the involvement of cytotoxic "reversed signaling" in the pathogenesis of autoimmune diseases.

A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.

Background The NORE1 protein was identified in a yeast two-hybrid screen as a Ras effector that binds Ras protein in a GTP-dependent manner. NORE1A is a growth and tumour suppressor that is inactivated in a variety of cancers. In transformed human cells, both full-length NORE1A protein and its effector domain alone (amino acids 191–363) are localized to microtubules and centrosomes. However, the mechanism by which NORE1A associates with these cytoskeletal elements is not known; furthermore, whether centrosomally-associated or microtubule-associated NORE1A suppresses tumour cell growth has not been yet established. Findings We have shown that purified NORE1A fails to bind to microtubules in vitro suggesting that other protein(s) mediate NORE1A-microtubule association. Using mass-spectrometry, we identified the Microtubule-Associated Protein 1B (MAP1B) and its homologue C19ORF5 as NORE1A interaction partners. Suppression of C19ORF5 expression by RNA interference (RNAi) and immunodepletion of C19ORF5 protein from cell extracts showed that binding of NORE1A to microtubules is not dependent on C19ORF5. Conversely, RNAi suppression of MAP1B revealed that MAP1B is required for association of NORE1A with microtubules. RNAi-mediated depletion of C19ORF5 or MAP1B did not prevent centrosomal localization of NORE1A. Moreover, the depletion of C19ORF5 or MAP1B did not prevent NORE1A's ability to suppress tumour cell growth. Conclusion The interaction of NORE1A with microtubules is mediated by MAP1B, but not C19ORF5 protein. Interaction of NORE1A with centrosomes is not dependent on C19ORF5 or MAP1B, and appears to involve a different mechanism independent of binding to microtubules. The NORE1A microtubular localization is not required for growth suppression.

NORE1A is a growth and tumor suppressor that is inactivated in a variety of cancers. NORE1A has been shown to bind to the active Ras oncogene product. However, the mechanism of NORE1A-induced growth arrest and tumor suppression remains unknown. Using anchorage-independent growth assays, we mapped the NORE1A effector domain (the minimal region of the protein responsible for its growth-suppressive effects) to the fragment containing the central and Ras association domains of NORE1A (amino acids 191-363). Expression of the NORE1A effector domain in A549 lung adenocarcinoma cells resulted in the selective inhibition of signal transduction through the ERK pathway. The full-length NORE1A (416 amino acids) and its fragments capable of growth suppression were localized to centrosomes and microtubules in normal and transformed human cells in a Ras-independent manner. A mutant that was deficient in binding to centrosomes and microtubules was also deficient in inducing cell cycle arrest. This suggests that cytoskeletal localization is required for growth-suppressive effects of NORE1A. Ras binding function was required for growth-suppressive effects of the full-length NORE1A but not for the growth-suppressive effects of the effector domain. Our studies suggest that association of NORE1A with cytoskeletal elements is essential for NORE1A-induced growth suppression and that the ERK pathway is a target for NORE1A growth-suppressive activities.

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated “striated muscle contraction” as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.