Patent Application
20140370079
1. Field of the Invention
The present application relates to a method of targeting RhoJ for suppressing tumor angiogenesis and inducing vascular disruption.
2. General Background and State of the Art
Vascular targeting therapies have been considered as one of the anti-cancer therapeutic options for the past decade. However, the survival benefit is usually only several months, depending on clinical conditions, because of intrinsic resistance and evasive mechanisms. Here, we show the potential of RhoJ blockade, through inhibition of angiogenesis and disruption of existing vessels in tumors, to be a powerful adjuvant option to complement and maximize the anti-cancer effects of conventional anti-angiogenic and vascular-disrupting agents. Our study provides a rationale for the development of specific inhibitors against RhoJ.
Tumor angiogenesis is a prerequisite for tumor progression (Ferrara and Alitalo, 1999; Hanahan and Folkman, 1996). The angiogenic switch is activated during tumor growth, and resulting tumor neovessels manage the O2 and nutrient requirements as well as the clearance of CO2 and metabolite in tumor tissue (Carmeliet and Jain, 2011; Hanahan and Folkman, 1996). Moreover, the tumor vasculature is one of main route of tumor cell metastasis to distant organs (Hanahan and Weinberg, 2011). Collectively, these observations imply that tumors cannot grow further and metastasize without sufficient blood supply. This inference has led to the development of various angiogenesis-inhibiting agents (AIAs) in the past decade (Ellis and Hicklin, 2008; Ferrara and Kerbel, 2005; Sennino and McDonald, 2012), many of which target vascular endothelial growth factor (VEGF) and its receptors and have proved to be effective in clinical practice (Carmeliet and Jain, 2011; Chung et al., 2010). In addition, ongoing drug development has focused on moderating other angiogenic pathways (Bono et al., 2013; Gerald et al., 2013; Koh et al., 2010; Sennino and McDonald, 2012; Tvorogov et al., 2010). Because current AIAs are inherently cytostatic and target newly growing tumor vasculature, they are more suited to tumor stabilization than to the regression of a bulky tumor (Ellis and Hicklin, 2008; Horsman and Siemann, 2006). Even after repeated cycles of AIA treatment, a substantial amount of preformed vasculature remains intact within the tumor. In addition, although it requires further investigation in clinics, several preclinical studies suggested that tumor cells could convert to a more aggressive phenotype with increased invasion and metastasis after the AIA treatment (Bergers and Hanahan, 2008; Casanovas et al., 2005; Ebos and Kerbel, 2011). Moreover, because VEGF and its receptors are expressed ubiquitously in normal tissues and in tumors, current AIAs produce adverse effects such as hypertension, proteinuria, and hemorrhage (Chen and Cleck, 2009; Kamba and McDonald, 2007). Therefore, it is important to better discern differences between tumor and normal vasculature in order to develop more selective and potent targeting strategies.
Rho GTPases have recently been discovered as fine-tuners of vascular morphogenesis and homeostasis (Bryan and d'Amore, 2007). Rho GTPases are considered as essential downstream targets of VEGF signaling in endothelial cells (ECs), and a well-controlled balance between different Rho GTPases governs almost all aspects of angiogenic processes such as EC migration, proliferation, extracellular matrix degradation, vascular morphogenesis, and vascular integrity (Beckers et al., 2010; Bryan and d'Amore, 2007; van der Meel et al., 2011). Although much remains to be unraveled about how different Rho GTPases are involved in angiogenesis and coordinate with each other, targeting Rho GTPases has become a promising strategy to enhance current anti-angiogenic treatment (van der Meel et al., 2011). One question to be answered is which Rho GTPase is the most promising anti-angiogenic target with high selectivity against tumor vasculature.
RhoJ is a Rho GTPase mainly expressed in ECs (Fukushima et al., 2011; Kaur et al., 2011; Takase et al., 2012; Yuan et al., 2011), and its expression is regulated by the endothelial transcription factor ERG in primary cultured human umbilical vein endothelial cells (HUVECs) (Yuan et al., 2011). Despite its vascular expression pattern, the importance of RhoJ in vascular biology is only beginning to emerge. A few recent papers have revealed that RhoJ is an important regulator of EC motility and tube morphogenesis in 3D matrices (Kaur et al., 2011; Yuan et al., 2011). During development, RhoJ is specifically expressed in the dorsal aorta and intersomitic vessels of mouse embryos as well as in the retinal vessels of the postnatal mouse (Fukushima et al., 2011; Kaur et al., 2011). RhoJ-deficient mice display delayed radial growth of retinal vasculature during postnatal development with increased vascular regression in the vascular front (Takase et al., 2012). Also, RhoJ-overexpressing mice attenuate the aberrant extraretinal vascular outgrowth in an oxygen-induced retinopathy model (Fukushima et al., 2011). Thus, RhoJ signaling primarily affects vessel remodeling via balancing neovessel formation and regression; however, the expression and function of RhoJ in tumor angiogenesis have not been elucidated thus far.
Because RhoJ is specifically expressed in ECs during development (Fukushima et al., 2011; Kaur et al., 2011; Leszczynska et al., 2011), we speculated that it is also expressed in the growing tumor vasculature. Here, we investigated the biological role and therapeutic relevance of targeting RhoJ in various solid tumor models.
Current anti-angiogenic therapy is limited by its cytostatic nature and systemic side effects. To address these limitations, we have unveiled the role of RhoJ, an endothelial-enriched Rho GTPase, during tumor progression. RhoJ blockade provides a double assault on tumor vessels by both inhibiting tumor angiogenesis and disrupting the preformed tumor vessels, through the activation of the RhoA-ROCK (Rho kinase) signaling pathway in tumor endothelial cells, consequently resulting in a functional failure of tumor vasculatures. Moreover, enhanced anti-cancer effects were observed when RhoJ blockade was employed in concert with a cytotoxic chemotherapeutic, angiogenesis-inhibiting agent or vascular-disrupting agent. These results identify RhoJ blockade as a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects.
The invention overcomes the above-mentioned problems, and provides a selective and effective therapeutic strategy for targeting tumor vasculature with minimal side effects.
In one aspect, the present invention is directed to a method of inhibiting tumor growth comprising contacting the tumor with a compound that inhibits activity of RhoJ protein. The tumor growth may occur in a subject, in which the subject may be a mammal and in particular a human being.
In another aspect, the present invention is directed to a method of inhibiting cancer metastasis in a subject comprising administering to the subject a compound that inhibits activity of RhoJ protein. The cancer metastasis may occur in a subject, in which the subject may be a mammal and in particular a human being.
In another aspect, the present invention is directed to a method of reducing tumor volume comprising contacting the tumor with a compound that inhibits activity of RhoJ protein. The tumor volume reduction may occur in a subject, in which the subject may be a mammal and in particular a human being.
In another aspect, the present invention is directed to a method of disrupting tumor vasculature comprising contacting the tumor with a compound that inhibits activity of RhoJ protein. The tumor vasculature disruption may occur in a subject, in which the subject may be a mammal and in particular a human being, and further wherein the tumor vasculature may be disrupted selectively.
In any of the above aspects, the compound may be an oligonucleotide complementary to a portion of RhoJ transcript, an antagonistic ligand of RhoJ, or a chemical compound that inhibits the activity of RhoJ.
In any of the above aspects, the inventive method may include further contacting the tumor with or administering to the subject, a compound that sequesters VEGF in combination with the compound that inhibits activity of RhoJ protein. The compound that sequesters VEGF may be preferably VEGF-trap.
In any of the above aspects, the inventive method may further include contacting the tumor with or administering to the subject, a vascular-disrupting agent (VDA) in combination with the compound that inhibits activity of RhoJ protein. The VDA may be preferably combretastatin-A4-phosphate (CA4P).
In any of the above aspects, the inventive method may further include contacting the tumor with or administering to the subject, a cytotoxic therapeutic agent in combination with the compound that inhibits activity of RhoJ protein. The cytotoxic therapeutic agent may be preferably cisplatin.
In any of the above aspects, the compound or agent may be included in a carrier.
In any of the above aspects, the carrier may be an aptide conjugated liposome.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein;
Table 1 shows characteristics of the patient cohort (n=216) as related to
In the present application, “a” and “an” are used to refer to both single and a plurality of objects.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field.
As used herein, “RhoJ blockade” or “inhibiting RhoJ activity” refers to the inhibition of RhoJ activity in an organism. The inhibition may occur by binding RhoJ with selective small molecules that nullify the RhoJ activity, or depleting RhoJ at the protein level with an anti-sense oligonucleotide such as an siRNA, or inhibiting a member in the RhoJ reaction cascades that results in reduction of RhoJ activity.
As used herein, “a chemical compound that inhibits the activity of RhoJ” refers to a chemotherapeutic agent that is specific to RhoJ. Such compounds may be produced by synthesizing chemicals and assaying for their activity against RhoJ activity.
As used herein, “disrupting tumor vascular integrity” refers to specifically and selectively destroying the blood vessels present in a tumor, but not normal blood vessels as is manifest as intratumoral hemorrhagic necrosis.
As used herein, “sequestering VEGF” refers to a molecule such as VEGF-trap, which is used to bind VEGF so that VEGF is neutralized and is not active.
As used herein, “vascular-disrupting agent” refers to an agent known in the art that disrupts pre-formed blood vessels, such as combretastatin-A4-phosphate.
As used herein, “cytotoxic therapeutic agent” refers to any agent that is toxic to a cell, and includes a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term includes radioactive isotopes, toxins such as small-molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. Preferably, the agent is a chemotherapy drug, such as cisplatin.
Known anti-cancer chemical compounds that are useful in the treatment of cancer exist, and may be used together with the inventive anti-RhoJ compounds. Examples of some of these compounds include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammal1 and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.
As used herein, administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
As used herein, “antagonist” refers to a ligand that tends to nullify the action of another molecule.
As used herein, “carriers” include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the pharmaceutically acceptable carrier is an aqueous pH buffered solution. Examples of pharmaceutically acceptable carriers include without limitation buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
Carrier as used in the present application includes “aptide”, which refer to a class of high-affinity peptides, which are typically conjugated to another entity such as drug-containing liposomes for cancer therapy. Such drug may include an oligonucleotide such as siRNA or a protein or a chemical compound.
As used herein, “consisting essentially of” when used in the context of a nucleic acid sequence or amino acid sequence refers to the sequence that is essential to carry out the intended function of the amino acid encoded by the nucleic acid.
As used herein, “effective amount” is an amount sufficient to effect beneficial or desired clinical or biochemical results. An effective amount can be administered one or more times. For purposes of this invention, an effective amount of an inhibitor compound is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
As used herein, “ligand” refers to any molecule or agent, or compound that specifically binds covalently or transiently to a molecule such as a polypeptide. When used in certain context, ligand may include antibody. In other context, “ligand” may refer to a molecule sought to be bound by another molecule with high affinity, such as in a ligand trap.
As used herein, “mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, and so on. Preferably, the mammal is human.
As used herein “pharmaceutically acceptable carrier and/or diluent” includes any and all solvents, dispersion media, coatings antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
As used herein, “subject” is a vertebrate, preferably a mammal, more preferably a human.
As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. “Palliating” a disease means that the extent and/or undesirable clinical manifestations of a disease state are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment.
As used herein, “vector”, “polynucleotide vector”, “construct” and “polynucleotide construct” are used interchangeably herein. A polynucleotide vector of this invention may be in any of several forms, including, but not limited to, RNA, DNA, RNA encapsulated in a retroviral coat, DNA encapsulated in an adenovirus coat, DNA packaged in another viral or viral-like form (such as herpes simplex, and adeno-structures, such as polyamides.
Role of RhoJ in the Regulation of Tumor Angiogenesis and Tumor Vascular Integrity
Here, we have demonstrated a critical role of RhoJ in the regulation of tumor angiogenesis and tumor vascular integrity. The phenotypic endpoints of RhoJ blockade are similar to those of blockade of another small GTPase, R-Ras; genetic disruption of R-Ras also severely impairs EC barrier function, resulting in disturbed tumor vasculature maturation (Sawada et al., 2012). Furthermore, it has recently been reported that RhoJ is strongly expressed in human cancers, being one of the top 10 genes of the common angiogenesis signature (Masiero et al., 2013). This correlates well with our data showing that high expression of RhoJ in colon cancer is a negative prognostic factor in these patients, further highlighting RhoJ as a clinically relevant therapeutic target in cancer.
RhoJ blockade displayed several advantages over current vascular targeting therapy, but the most superior advantage is its “double assault” on tumor vessels. Vascular targeting agents developed during the past decade are commonly classified as either AIAs or VDAs. AIAs mainly suppress the formation of tumor neovessels and induce tumor vessel normalization, whereas VDAs directly disrupt preformed tumor vessels and shut down blood flow, finally resulting in massive tumor necrosis and hemorrhage (Tozer et al., 2005). AIAs are particularly effective in the peritumoral regions of newly progressing tumors where new tumor vessels are robustly developing, while VDAs are most effective in the intratumoral regions of established tumor where preformed immature vessels are abundant (Horsman and Siemann, 2006; Siemann, 2011). RhoJ blockade encompasses both aspects of AIAs and VDAs and offers an effective strategy for targeting tumor vasculatures: It simultaneously impedes the formation of tumor neovessel and disrupts the pre-established tumor vessel network. Through this “double assault” on tumor vasculature, RhoJ blockade markedly inhibited blood flow to tumor cells and displayed a convincing anti-cancer and anti-metastatic effect.
In addition, RhoJ blockade compensates for and augments other anti-cancer therapies. The combination therapy of RhoJ blockade and the conventional chemotherapeutic drug, cisplatin, proved to be very effective in delaying tumor progression. As is previously known, the intratumoral core of tumors is resistant to conventional anti-cancer therapies (Trédan et al., 2007; Wachsberger et al., 2003), because anti-cancer drug delivery to this core is limited and inefficient due to the immature tumor vessels and increased interstitial pressure (Fukumura and Jain, 2007). Additionally, tumor cells in the intratumoral core have an intrinsic resistance to chemotherapy because they proliferate slowly and the growth fraction is small (Trédan et al., 2007). Intriguingly, RhoJ blockade preferentially induces vascular shutdown in intratumoral regions, resulting in necrosis of the tumor cells. By combining cisplatin and the RhoJ blockade, both of which exert distinctive modes of action, we achieved a comparatively enhanced anti-tumor and anti-metastatic effect, which suggests the potential of RhoJ blockade as an adjuvant for conventional chemotherapies. Moreover, the combination of RhoJ blockade with VDAs also showed an enhanced anti-tumor efficacy. Most VDAs target the tubulin cytoskeleton of tumor ECs directly and induce activation of RhoA-ROCK signaling in tumor ECs, resulting in the rapid and selective disruption of the preformed tumor vessels (Siemann, 2011). However, in spite of promising preclinical results, they failed to show efficacy in clinical trials (Baguley and McKeage, 2012). The major drawback of VDAs is that they mainly target the intratumoral core, leaving the remaining peripheral viable rim to regrow and even acquire resistance to VDAs (Horsman and Siemann, 2006; Tozer et al., 2005). In contrast, RhoJ blockade in the present study exerted its anti-tumor effect through inhibition of neovessel formation in both the peri- and intratumoral regions and also enhanced shutdown of pre-existing tumor vessels in the intratumoral regions. Furthermore, we found that RhoJ blockade shares its action mechanism with VDAs, also activating the RhoA-ROCK signaling pathway. In this regard, it is logical to speculate that RhoJ blockade may be complementary to current VDA therapies. Indeed, we confirmed that RhoJ blockade could overcome the resistance acquired from VDA monotherapies, such as CA4P, with regard to tumor growth and progression.
Previous studies have found that VEGF-A stimulation regulates the activity of various Rho GTPases, such Cdc42, Rac 1, and RhoA, whereas interactions among various Rho GTPases are poorly understood (Beckers et al., 2010; Bryan and d'Amore, 2007; Schiller, 2006). Blocking the RhoJ pathway over a prolonged period raises the possibility of compensatory activation of other Rho GTPases in tumor vessels, especially by Cdc42 and Racl, which share common downstream effector molecules with RhoJ (Leszczynska et al., 2011). From this perspective, the concurrent inhibition of RhoJ signaling and VEGF-A signaling could be an attractive therapeutic strategy not only by enhancing current AIA therapy but also by maximizing the vascular-disrupting effect of the RhoJ blockade. Indeed, our findings strongly support this possibility. The combination of RhoJ blockade and VEGF decoy receptor, VEGF-trap, showed comparatively potent anti-angiogenic activity in both peri- and intratumoral areas of the LLC tumor, which are known to be resistant to conventional AIA therapies (Shojaei et al., 2007). Another possible benefit from this combination is that RhoJ blockade may maintain and maximize responses to the AIA therapies. It is known that tumor vessels regrow alongside the ghost tracks of remnant BM after cessation or during the resting period of AIA treatment (Mancuso et al., 2006). Intriguingly, we observed a severe loss of BM in RhoJ-deficient tumor vessels, indicating that concurrent RhoJ blockade might abolish remnant BM in concert with AIAs and prevent tumor vessel from regrowth, finally resulting in a sustained response to the AIA therapies.
An additional advantage of RhoJ blockade is that it selectively targets tumor vessels with minimal systemic side effects. Current AIAs influence normal vessels as well because their main targets, VEGF-A and its receptors, are expressed ubiquitously. Therefore, they induce systemic side effects such as hemorrhage, hypertension, proteinuria, and delayed wound healing (Chen and Cleck, 2009; Kamba and McDonald, 2007). On the other hand, RhoJ expression is very specific to pathologic conditions, especially in tumor tissues, while being rarely expressed in organs under normal physiologic conditions; the global deletion of RhoJ does not induce gross abnormalities and lethality. However, our results indicate that RhoJ plays a positive angiogenic role during wound healing, and this could be an unavoidable side-effect of a putative RhoJ inhibitor.
Finally, RhoJ is a feasible target for clinical drug development. We could therapeutically target RhoJ in tumor tissues through an in vivo siRNA delivery system. Using the APTEDB-LS complex as a carrier, which has high specificity against tumor tissues (Kim et al., 2012), we effectively delivered siRhoJ into tumor tissues and significantly delayed tumor growth and metastasis, especially in concert with VEGF-trap. Consequently, we established a way to clinically inhibit RhoJ.
In conclusion, our evidence shows that RhoJ is a promising selective target in the tumor vasculature that governs the processes of tumor angiogenesis and vascular integrity. The distinguishing characteristics of RhoJ blockade provide a strategy for overcoming the limitations of current vascular targeting therapies in patients with advanced cancer. Further development of specific RhoJ inhibitors is needed to ascertain their efficacy and safety in clinical settings.
In one embodiment, the present invention relates to anti-cancer treatment
The formulation of therapeutic compounds is generally known in the art and reference can conveniently be made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., USA. For example, from about 0.05 μg to about 20 mg per kilogram of body weight per day may be administered. Dosage regime may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intra nasal, intradermal or suppository routes or implanting (eg using slow release molecules by the intraperitoneal route or by using cells e.g. monocytes or dendrite cells sensitised in vitro and adoptively transferred to the recipient). Depending on the route of administration, the peptide may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate said ingredients.
The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, theomersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterile active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
When the peptides or chemicals or oligonucleotides are suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.
The tablets, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
Delivery Systems
Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody or a peptide of the invention, care must be taken to use materials to which the protein does not absorb. In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome. In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The following examples are offered by way of illustration of the present invention, and not by way of limitation.
Animal care and experimental procedures were performed under the approval (KA2011-17) from the Animal Care Committee of KAIST. Specific pathogen-free (SPF) C57BL/6J and MMTV-PyMT transgenic mice (FVB/N) were purchased from Jackson Laboratory (Bar Harbor, Me.). RhojGFP/GFP and Rhoffl mice, and Cdh5(PAC)-CreERT2 mice (Wang et al., 2010b) were transferred and bred in our SPF facilities. To deplete Rhoj in MMTV-PyMT tumors, RhojGFP/GFP female mice were intercrossed with MMTV-PyMT male mice. To deplete Rhoj specifically in ECs, Rhoffl/fl mice were intercrossed with Cdh5(PAC)-CreERT2 mice. All mice were fed with ad libitum access to standard diet (PMI Lab diet) and water. All mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg of ketamine and 12 mg/kg of xylazine) before being sacrificed.
LLC and B16F10 melanoma cells were obtained from American Type Culture Collection. To generate implantation tumor models, suspensions of tumor cells (1×106 cells in 100 μl) were SC injected into the dorsal flank of 8 to 10 weeks old mice. Tumor volumes were measured at given time points. Tumor volume was calculated according to the formula, 0.5×A×B2, where A is the largest diameter of a tumor and B is its perpendicular diameter. Tumor growth rate is defined as increased tumor volume relative to 2 days before. Indicated days later, the mice were anesthetized and tissues were harvested for further analyses. Tamoxifen (4 mg/kg, 4 times every 2 days, Sigma-Aldrich) was IP injected into Cdh5(PAC)-CreERT2;Rhorfl/fl mice starting from the day before tumor implantation or after the tumor volume had exceeded 300 mm3. Cisplatin (10 mg/kg, every 7 days, Sigma-Aldrich) is IP injected for cytotoxic chemotherapy when tumor volume exceeded 100 mm3. VEGF-trap (25 mg/kg, indicated schedule) is SC injected as an AIA therapy. CA4P (50 mg/kg, every 2 days, Sigma-Aldrich) was IP injected as a VDA therapy. As a control, equal amounts of Fc or PBS was injected in the same manner. To knock down RhoJ in vivo, control or RhoJ siRNA (2 mg/kg, indicated schedule), which were encapsulated into aptEDB-LS complexes, were IV injected into tumor-bearing mice.
All human samples were collected by the tissue bank of Severance Hospital, Seoul, Korea, with the informed consents from the donors, following the bioethics and safety regulations. All procedures regarding human samples were performed with the approval of institutional review board (KH2013-02).
To generate Rhoj mutant mice, a targeting construct was assembled, which contains a loxP-mouse Rhoj cDNA-pA-loxP-EGFP-pA-FRT-SV40 early promoter-Neo-pA-FRT cassette (Uesaka et al., 2007) flanked by 8-kb 5′ and 3-kb 3′ arms which were generated by PCR using a C57BL/6-derived BAC clone RP23-280114 (BACPAC Resource Center) as a template (
To produce recombinant proteins, dimeric-Fc (Fc) and VEGF-trap, stable CHO cell lines that secrete these recombinant proteins were used as previously described (Koh et al., 2010). Recombinant proteins in supernatant were purified by column chromatography with Protein A agarose gel (Oncogene) using acid elution. After purification, the recombinant proteins were quantified using the Bradford assay and confirmed by Coomassie blue staining after SDS-PAGE.
The skin wound healing assay was conducted as described previously (Zhou et al., 2004). Two round 5-mm full-thickness punch wounds were made on the dorsal skin of 8-week old mice using a biopsy punch (Miltex). The progression of wound healing was observed and photographed every 2 days over the following 6 days. At day 6 after creating the wound, the wound tissue was harvested for histologic analyses.
To analyze the influence of RhoJ expression in human cancer patients, we acquired the mRNA sequencing (RNASeq) data from The Cancer Genome Atlas (TCGA) database (http://cancergenome.nih.gov). Of the 438 colon cancer patients registered in TCGA, 405 patients had the RNASeq data available. Among various RNASeq platforms, we chose Illumina HiSeq V2 (RNASeqV2), the platform with which the largest number of patients were included (n=216). MapSplice (Wang et al., 2010a) was used for alignment and RNASeq by Expectation Maximization (RSEM) (Li and Dewey, 2011) was used to determine RhoJ expression levels. Of the 216 colon cancer patients, the RNA SeqV2 level 3 data were used to obtain the normalized RhoJ expression levels and clinical data were used to obtain various clinical attributes which were summarized in Table 1. The survival attribute was computed from ‘days_to_last_followup’ or ‘days_to_last_known_alive’ if the patients are still alive, and ‘days_to_death’ if the patients are dead. The clinical outcome attribute indicates whether the patient is dead (1) or not (0). Each patient in the TCGA database has their own ID so we are able to map every tissue sample to the corresponding patient. All 216 colon cancer patients were divided into RhoJ-high (n=78) or RhoJ-low (n=138) groups in which the cut-off value was the average RhoJ expression level of all patients. Every processing related to the TCGA database was done with Python software.
For hematoxylin and eosin (H&E) staining, tumors and indicated organs were fixed overnight in 4% paraformaldehyde (PFA). After tissue processing using standard procedures, samples were embedded in paraffin and cut into 3-1 μm sections followed by H&E staining. For immunofluorescence studies, samples were fixed in 1% PFA, dehydrated in 20% sucrose solution overnight, and embedded in tissue freezing medium (Leica). Frozen blocks were cut into 50-1 μm sections. Samples were blocked with 5% goat (or donkey) serum in PBST (0.03% Trition X-100 in PBS) and then incubated for 3 hr at room temperature (RT) with the following primary antibodies: anti-GFP (rabbit polyclonal, Millipore), anti-CD31 (hamster, clone 2H8, Millipore), anti-RhoJ (mouse, clone 1E4, Novus), anti-RhoJ (mouse, clone 1D7, OriGene), FITC-conjugated anti-c′-SMA (mouse, clone 1A4, Sigma-Aldrich), anti-VE-cadherin (rat, clone 11D4.1, BD Phamingen), anti-VE-cadherin (rabbit, clone D87F2, Cell Signaling), anti-PDGFRβ (rat, eBioscience), anti-Ter119 (rat, clone TER-119, eBioscience), anti-caspase-3 (rabbit polyclonal, R&D systems), anti-LYVE-1 (rabbit polyclonal, Angiobio), anti-pan-cytokeratin (mouse, clone AE1/AE3, Abcam), anti-melanin-A (rabbit polyclonal, Abcam), anti-collagen type IV (rabbit polyclonal, Cosmo Bio), or anti-cisplatin modified DNA (rat monoclonal, Abcam). After several washes, the samples were incubated for 2 hr at RT with the following secondary antibodies: FITC-, Cy3-, or Cy5-conjugated anti-hamster IgG (Jackson ImmunoResearch), FITC- or Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch), Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch), or Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch). Goat Fab fragment anti-mouse IgG (Jackson ImmunoResearch) was used to block endogenous mouse IgG to use mouse antibody on mouse tissues. F-actin was stained with acti-stain 555 phalloidin (Cytoskeleton). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Then the samples were mounted with fluorescent mounting medium (DAKO) and immunofluorescent images were acquired using a Zeiss LSM510 confocal microscope (Carl Zeiss). To detect the hypoxic areas in the tumors, Hypoxyprobe-1™ (60 mg/kg, solid pimonidazole hydrochloride, Natural Pharmacia International) was IV injected 90 min before perfusion-fixation. The tumors were then harvested, sectioned, and stained with FITC-conjugated anti-Hypoxyprobe antibody.
At indicated days, tumor vessel leakage was analyzed after IV injection of 100 μl of FITC or rhodamine-conjugated dextran (25 mg/ml, 70 kDa, Sigma-Aldrich) 30 min before sacrifice. For vascular perfusion studies, 100 μl of DyLight® 594-conjugated tomato lectin (1 mg/ml, Vector laboratory) was IV injected 10 min before sacrifice. Mice were anesthetized and perfused by intracardiac injection of 1% PFA to remove circulating dextran and lectin.
Density measurement of blood vessels, metastasis, hemorrhagic area, leakage area, and perfusion area were performed with ImageJ software (http://rsb.info.nih.gov/ij). For blood vessel density, CD31+ area per random 0.42 mm2 areas was measured in the peri- and intratumoral regions. To determine the amount of blood vessels that express RhoJ, GFP+&CD31+ area per CD31+ area in five random 0.42 mm2 areas were calculated. In addition, to find the percentage of CD31+ endothelial cells within RhoJ-GFP expressing cells, the GFP+&CD31+ area per total GFP+ area was also calculated. The measurements of metastasized cytokeratin+ cells in the lymph nodes were made on the total mid-section area. The area of cytokeratin+ fluorescence was presented as % per total sectioned area of lymph node. The measurements of hemorrhagic, necrotic and viable areas of tumors were made on the total mid-section area. The extent of hemorrhage was measured as a % of Ter-119+ area per random 0.24 mm2 areas. Vascular leakage was quantified as the dextran+ area % per random 0.42 mm2. Vascular perfusion area was calculated as the percentage of lectin+ area divided by CD31+ area in random 0.42 mm2 regions. As for the quantification of lung metastasis, only the tumor colonies >100 μm in diameter were enumerated. Coverage of α-SMA+ or PDGFRβ+ mural cells and collagen-IV+ BM was calculated as the percentage of corresponding fluorescent positive length along the CD31+ vessels in random 0.42 mm2 intratumoral regions. The numbers of vascular sprouts (>10 μm in length from the site of protrusion to the tip) were measured in the random 1 mm2 peri- and intratumoral areas. The extent of cisplatin retention was measured as a % of cisplatin-modified DNA+ area per random 0.24 mm2 areas. The photographs of remaining wound area were quantified using ImageJ software. The granulation area was measured as the cross-sectional granulation area (mm2) per total wound area. The calculation was performed using the middlemost section of the wound stained with H&E. All measurements were performed at least five different fields per mice.
Tumor samples and lungs were harvested, chopped into small pieces, and digested into single cell suspension by incubating in digestion buffer (0.1% collagenase type 4 (Worthington) and 3 U/ml DNase I (Worthington)) for 1 hr at 37° C. The digested cells were filtered with a 40 μm nylon mesh to remove cell clumps. Cells were incubated for 10 min with the following antibodies in FACS buffer (5% bovine serum in PBS): PE-conjugated anti-mouse CD31 (rat, clone MEC13.3, eBioscience) and APC-conjugated anti-mouse CD45 (rat, clone 30-F11, eBioscience) antibodies. After several washes, the cells were analyzed and sorted by FACS Aria II (Beckton Dickinson). The purity of the sorted cells was at least 95%. Dead cells were excluded using 7-aminoactinomycin D (7-AAD, Invitrogen).
Total RNA was extracted from cultured cells and purified cells from tumors using RNeasy plus micro kit (Qiagen) according to the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA using SuperScript® II Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed with indicated primer pairs listed below by using fast SYBR® green master mix (Roche) and CFX 96 Real-Time PCR Detection System (Bio-Rad™). The real-time PCR data were analyzed with CFX Manager Software (Bio-Rad™).
HUVECs were purchased from Lonza, cultured in endothelial growth medium (EGM-2, Lonza) and incubated in a humidified atmosphere with 5% CO2 at 37° C. The cells used were between passages 3 to 8. Transfections of siRNA duplexes into HUVECs were performed using Lipofectamine® RNAiMAX (Invitrogen) at a final concentration of 40 nM according to the manufacturer's protocol.
WGIIRLEQ (SEQ ID NO:7) was screened by phage-display technology and was synthesized (Anygen Corp) (Kim et al., 2012). The conjugation of APTEDB and Mal-PEG2000-DSPE was carried out for 12 hr in RT, in which the molar ratio of was 1:2. The conjugation efficiency was then confirmed using a MALDI-TOF. For the preparation of anionic liposomes, POPC:Chol:POPG (molar ratio, 4:3:3; +/N/− charge ratio, 6:1:6) was added to make a lipid film as described previously (Saw et al., 2010). For APTEDB targeting liposomes, 2.5 wt % of APTEDB-PEG2000-DSPE were added to the original liposome. For Cy5.5 labeled liposomes, thiol-modified Cy5.5 (Lumiprobe) was conjugated with Mal-PEG2000-DSPE with the same protocol described above. Finally, to encapsulate RhoJ siRNA into the APTEDB-liposome core, RhoJ siRNA was first complexed with 9R at 1:4 N/P ratio in HBG 5% buffer as described previously (Saw et al., 2010). 30 minutes after complexation, the complex was then added into the lipid film. To verify the successful delivery of APTEDB-liposome into tumors, near-infrared images were taken at the indicated time points using an IVIS imaging machine (Xenogen). The effective knockdown of RhoJ in tumor tissue was confirmed using Immunoblotting.
For EC migration assay, HUVECs were plated on a cell culture plate at 20% confluency. After 12 hr, migration of HUVECs was recorded as time-lapse movies. A Chamide magnetic chamber (Live Cell Instrument, Seoul, Korea) was kept at 37° C. and 5% CO2 during experiment. An Axiovert 200M microscope (Carl Zeiss) equipped with an AxioCam MRm (Carl Zeiss) was used. Phase contrast images were acquired every 3 min for 6 hr. Migration patterns and speeds of HUVECs were analyzed by ImageJ software.
To evaluate the directional migration and angiogenic sprouting of ECs, we applied a 3D microfluidics system which we modified from the previously employed device (
For matrigel tube formation assay, growth factor reduced Matrigel (BD bioscience) was thawed overnight at 4° C. The Matrigel was allowed to solidify on a 4-well culture dishes at 37° C. for 30 min. Cells were harvested and seeded at a density of 2×104 cells/well in growth media. Cells were then incubated at 37° C. for a further 12 hr. Tube formation was observed by taking pictures using a Leica DM IL microscope. The matrigel assays were quantified by counting the number of nodes and tubules from five different fields for each condition.
For in vitro vascular permeability assay, HUVECs were cultured on collagen-coated 1.0 μm-size pore insert (Millipore). After starvation for 12 hr, the cells were treated with or without VEGF (50 ng/ml) or CA4P (20 nM) for 2 hr and was then incubated with 70 kDa FITC-Dextran for 20 min. Each solution in plate wells were read with a Victor X2 multilabel plate reader.
RhoA activities were determined using a RhoA activation assay kit (BK036, Cytoskeleton). HUVECs were cultured at 40% confluence, starved overnight, and treated with VEGF-A (50 ng/ml) for 10 min. Cells were lysed and the cell lysates were incubated with the rhotekin beads (50 μg/sample) for 1 h at 4° C., washed two times, and eluted with Laemmli sample buffer. Bound RhoA, which is an active form of RhoA, was analyzed by SDS-PAGE separation followed by immunoblotting with an anti-RhoA antibody (ARH03, Cyto skeleton). The amount of total RhoA was also analyzed by immunoblotting using the same antibody in order to normalize the relative activity of RhoA.
ROCK activities were determined using a ROCK activity assay kit (CSA001, Millipore). HUVECs were lysed and cell lysates were incubated for 1 hr at room temperature in 96-well plates pre-coated with recombinant MYPT1, which contain Thr696 residue that can be phosphorylated by active ROCK. The plates were washed and incubated with anti-phospho-MYPT1 (Thr696) antibody followed by incubation with HRP-conjugated secondary antibody and HRP substrate reagent. The relative amount of active ROCK was measured by a microplate reader at 450 nm (Bio-Rad).
At the indicated time, tumor tissues or cultured HUVECs were homogenized in ice-cold lysis buffer containing a protease inhibitor cocktail (Roche). Each protein was separated with SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with the following primary antibodies in blocking buffer overnight at 4° C.: RhoJ (mouse, clone 1D4, Novus), ROCK1 (mouse, clone G-6, Santacruz), pMLC (rabbit polyclonal, Cell signaling), MLC (rabbit polyclonal, Cell signaling), RhoA (mouse monoclonal, Cytoskeleton), GAPDH (rabbit polyclonal, Santacruz), and β-actin (rabbit polyclonal, Sigma). Membranes were then incubated with HRP-conjugated secondary antibodies for 2 hr at RT. Chemiluminescent signals were developed with HRP substrate (Millipore) and detected with a LAS-1000 mini (Fuji film).
HUVECs were cultured on glass coverslips coated with 0.1% gelatin for overnight. Cells were fixed with 1% PFA and permeablized with ice cold 0.3% PBST for 5 min and blocked in 5% goat serum in 0.1% PBST for 1 hr at room temperature. Samples were incubated with VE-cadherin antibody (rabbit, clone D87F2, Cell Signaling) for 3 hr at room temperature. After several washes, Cells were incubated for 2 hr with the FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch). Nuclei were stained with DAPI (Invitrogen) and F-actin was stained with Acti-stain™ 488 Phalloidin (Cytoskeleton). Then the cells were mounted and analyzed using a LSM 510 confocal microscope (Zeiss).
Tissues (LLC tumor, heart, lung, liver, and kidney) and HUVECs were fixed with 2.5% glutaraldehyde in PBS overnight and washed with cacodylate buffer (0.1 M) containing 0.1% CaCl2. Samples were post-fixed for 2 hr with 1% OsO4 in cacodylate buffer (pH 7.2) and washed with cold distilled water. Dehydration was performed with ethanol series and propylene oxide. Samples were embedded in Embed-812, resin polymerized, sectioned, and mounted on a formvar-coated slot grid. After staining with 4% uranyl acetate and lead citrate, sample images were acquired with a Tecnai G2 Spirit Twin transmission electron microscope (FEI).
Values are presented as mean±standard deviation. Statistical differences between means were determined by unpaired Student t-test or analysis of variance with one-way followed by the Student-Newman-Keuls test. Chi-square test was used to analyze discrete variables. The survival curve was evaluated using the Kaplan-Meier method, and statistical differences were analyzed using the log-rank test. Bivariate correlation was evaluated by the two-tailed Pearson test. Statistical significance was set at p<0.05.
List of Primer Sets for Quantitative Real-Time PCR
List of siRNA Sequences
To unveil the role of RhoJ in tumor progression, we generated RhOjGFP/GFP (RhoJ-KO) mice, in which Rhoj is knocked out by replacing its exon 1 with GFP; with this construct, GFP is expressed instead of Rhoj under the transcriptional control of the Rhoj promoter (
To examine the relevance of RhoJ in human tumor angiogenesis, we assessed RhoJ expression in human tissues and confirmed that RhoJ is highly expressed in the tumor vessels of colon adenocarcinomas (7 of 12 samples) but is undetectable in normal colon tissues (0 of 10 samples) (
Taking the advantage that RhoJ-KO mice grew to adulthood normally, we used RhoJ-KO mice to address the role of RhoJ during tumor progression. We employed the LLC tumor model by subcutaneously (SC) injecting LLC cells into RhoJ-WT and KO mice. At 3 weeks after implantation, compared to WT mice, RhoJ-KO mice showed a 55% reduced tumor growth (
Tumor vasculature consists of malformed, disintegrated, leaky and highly branched vessels that continuously undergo vascular remodeling (McDonald and Baluk, 2002; Siemann, 2011; Trédan et al., 2007). Because RhoJ-KO mice displayed increased intratumoral hemorrhage compared to RhoJ-WT mice, we further investigated the role of RhoJ in vascular integrity and function. Interestingly, LLC tumor of RhoJ-KO mice had more disrupted tumor vessels (
To determine whether RhoJ deletion affects tumor progression broadly, we also evaluated the melanoma model by SC implantation of B16F10 cells into RhoJ-WT and KO mice. Consistent with the findings observed in LLC tumors, tumor growth was delayed by 52% in RhoJ-KO mice compared to RhoJ-WT mice (
As for the spontaneous tumor model, MMTV-PyMT mice were mated with RhojGFP/+ mice to generate MMTV-PyMT;Rhoj+/+ mice (P/RhoJ-WT) and MMTV-PyMT;RhojGFP/GFP mice (P/RhoJ-KO). At 14 weeks of age, P/RhoJ-KO showed reduced development of spontaneous mammary tumor nodules compared to P/RhoJ-WT (
We also evaluated the role of RhoJ in wound healing using a punch-wound healing model. Like tumor vessels, the blood vessels in the granulation area of wounds displayed high RhoJ expression (
To ascertain the role of RhoJ in tumor ECs during tumor angiogenesis, we generated inducible EC-specific RhoJ loss-of-function mice (RhoJ-KOEC) by mating Rhojfl/fl with Cdh5(PAC)-CreERT2 (Wang et al., 2010), in which the Rhoj allele was efficiently deleted in the ECs upon tamoxifen administration (
To determine the role of RhoJ in in vitro angiogenesis and vascular leakage, HUVECs transfected with either RhoJ siRNA (siJ-ECs) or control siRNA (siC-ECs) were used. To exclude the off-target effects, 5 independent RhoJ siRNA were designed, and 3 RhoJ siRNAs with the best performance (named J0, J1, and J2) was chosen for further experiments (
We next questioned whether RhoJ has any role in maintaining EC integrity because various Rho GTPases are also involved in endothelial integrity (Beckers et al., 2010; Bryan and d'Amore, 2007). To answer this question, an in vitro vascular permeability assay was applied to examine the changes in EC paracellular integrity (
To confirm the effect of RhoJ deletion in concert with conventional chemotherapeutic drugs, cisplatin (10 mg/kg) was intraperitoneally (IP) injected into RhoJ-KO mice once every week starting when tumor volume exceeded 100 mm3. Cisplatin significantly delayed LLC tumor growth by 90% in RhoJ-KO mice compared to a 64% decrease in RhoJ-WT mice (
Many Rho GTPases are activated by VEGF-A and share their common downstream effector molecules (Beckers et al., 2010; Schiller, 2006). Therefore, there is a possibility that VEGF-A-driven activation of other Rho GTPases may partially compensate for the effects of RhoJ ablation, limiting the anti-tumor effects of the RhoJ blockade. To resolve this potential problem and maximize the anti-tumor effect, we investigated the effect of VEGF-A blockade in the tumor progression of RhoJ-WT and KO mice. Administration of VEGF-trap (25 mg/kg) delayed LLC tumor growth by 88% in RhoJ-KO mice compared to a 47% decrease in RhoJ-WT mice (
Next, to establish a method for therapeutic blockade of RhoJ, a tumor-targeted siRNA delivery system (Kim et al., 2012) was employed. The aptide was designed and used according to a previous protocol (
VDAs are known to disrupt established tumor vessels by directly targeting the cytoskeletons of ECs (Siemann, 2011). Because RhoJ blockade is comparable to VDAs in inducing tumor vascular disruption, we speculated that RhoJ blockade might have an enhancing effect with VDAs, such as CA4P. The in vitro tube formation assay revealed that RhoJ knockdown in concert with CA4P (20 nM) treatment profoundly inhibited EC tube formation, inducing almost complete disruption, compared to single treatment with either CA4P or RhoJ siRNA (
All of the references cited herein are incorporated by reference in their entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein.
| Number | Date | Country | |
|---|---|---|---|
| 61835900 | Jun 2013 | US |
