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Cellular Biology and Urology


1. Antisense Research

The laboratory has developed (and patented) antisense oligonucleotides (oligos) which inhibit growth of prostate, breast and glioblastoma tumor cells, in both in vitro and in vivo models. Original formulations of these oligos consisted of linear sequences of nucleotide bases, synthesized complementary to mRNA encoding transforming growth factor-alpha (TGF-α) and its binding site the epidermal growth factor receptor (EGFR). These oligos work by hybridizing to mRNA, enhancing their destruction by RNAse, and result in translation inhibition of targeted proteins. Although antisense technology has been developing since 1978, most oligo studies have employed monospecific oligos which target only a single mRNA (usually about the AUG initiation codon).

While hundreds of antisense oligos sequences have been shown to significantly inhibit tumor cell growth, we have taken two different approaches to enhance their activity. The first is by combining oligo treatment either in combination or in a sequential pattern of administration with traditional chemotherapeutic agents. Chemotherapeutics we have evaluated include Cytoxan, Taxol, DES, mitoxantrone, and three different platinates. Our results from these studies have identified several combinations or sequences of administration which significantly enhance oligo activity. The second approach to enhance activity is to develop a new type of antisense compound. Previous types have all been “monospecific” and target only a single complementary mRNA binding site. We have developed the concept of bispecific oligos which target, within a single linear sequence, two different mRNA binding sites. The first bispecifics we evaluated were similar in length to our original compounds and were similarly modified to prevent nuclease degradation. They targeted both TGF-α and EGFR on a single linear molecule. In order to limit their length (and to aid in both delivery and in cellular uptake) their overall sequence has been truncated, and as a result each region is half their original length. The first bispecific binding sequences are still centered about the complementary mRNA AUG initiation codon. For each pair of targeted sequences we evaluated two different oligos, each having both binding specificities, but in alternative 5’ to 3’ tandem orientations. These variations could have differences in activity based upon tertiary structure and intramolecular binding. We have also studied such intramolecular binding within individual oligos by complementary bases using computer models, as demonstrated below. Further study could identify structures which enhance activity through specific conformation about their binding sites and by the addition of other bases we could theoretically form such structures in those lacking them. These new types of bispecific oligos have also been combined with traditional chemotherapeutics in order to further enhance their activity.

The first study utilized bispecifics which targeted two proteins associated with regulation of a single autocrine loop. Since cancer growth is influenced by many influences, it’s likely that bispecifics which target proteins associated with more than one pathway may be even more effective. Therefore an additional bispecific pair has been similarly constructed to target EGFR and the anti-apoptotic protein bcl-2.

Cellular

The above figure represents the MR42 bispecific oligo. Its possesses an extensive region of anti-parallel complementary binding between bases which could influence its activity by either availability or recognition of its binding sites. In the 5’ to 3’ direction a CAT sequence can be seen at the 3’ end of the 18 base (mer) sequence complementary to mRNA encoding bcl-2 (upper left in top loop). A second CAT sequence can also be seen in the center of the 19 mer binding site specific for mRNA encoding EGFR (3’ end of molecule).

Bispecific oligos were be tested alone and with chemotherapeutics against both prostate (PC-3, LNCaP) and breast cancer (UACC 897) cell lines. For breast cancer studies additional agents would include herceptin and tamoxifin.

Abstract of initial results

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2) are efficacious against PC-3 and LNCaP prostate tumors when evaluated in both in vitro and in vivo models. To enhance their activity, and also to introduce a significantly different type of multifunctional agent into this field, “bispecific” oligos were constructed containing truncated sequences (derived from MR1 and MR2) recognizing both TGF-α and EGFR mRNA internal binding sites, located about their respective AUG initiation codons. Two bispecifics were constructed having complementary sequences for both TGF-α and EGFR mRNA, but differing in their 5’ to 3’ tandem orientation (TGF-α/EGFR [MR12] and EGFR/TGF-α [MR21] sequences); and a second tandem pair having complementary sequences for EGFR and bcl-2 (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]. These bispecifics were tested in vitro against PC-3 and LNCaP prostate tumor cells, with comparisons made to the original monospecific oligos from which they were derived.

The purpose of this study was: 1) To validate the concept that these newly developed bispecific oligos have antitumor activity; 2) to identify dominant binding sequences which can be used to design other bispecifics which target additional tumor regulatory pathways; 3) to identify differences in effectiveness dependent upon binding site orientation; 4) to determine whether bispecifics are more effective if targeting one vs two different biochemical pathways.

Comparisons were made initially between oligos utilizing PC-3 or LNCaP cells incubated for 2 days with the agents followed by an additional 2 days in their absence. The first experiment demonstrated that the bispecific TGF-α/EGFR [MR12] and EGFR/TGF-α [MR21] oligos are at least as effective as their monospecific counterparts and the bispecific with the MR21 orientation is notably more effective than the MR1 monospecific by 64% (p = 0.014 by student t-test and p = 068 by the more stringent Mann Whitney U test). It also suggested that the sequence directed against EGFR contributed most to bispecific activity, particularly when inserted 5’ to the TGF-α binding sequence (MR21 orientation).

In the next series of experiments two new bispecific oligos were constructed (each containing the dominant EGFR activity) which were directed towards mRNA encoding proteins associated, for the first time, towards two different biochemical processes. MR24 with the EGFR complementary site at the 5’ end of the 37 mer oligo, followed by the bcl-2 complementary binding site at the 3’ end; and MR42 in the opposite orientation. MR1, MR2, MR4, MR12, MR21, MR24 and MR42 (1X and 2X in concentration) were cultured with cells as described above and compared to lipofectin containing controls. Each oligo addition significantly inhibited growth of PC-3 cells. MR42 was the most effective and significantly better than MR1 (p = 0.128), MR2 (p = 0.021), MR4 (p = 0.0002) and MR12 (p = 0.0032). 2X MR24 and 2X MR42 were better than their 1X concentration counterparts, but the differences were not significant.

In two similar experiments MR1, MR2, MR4, MR12, MR21, MR24 and MR42 were cultured with LNCaP cells and compared to lipofectin containing controls. Each oligo significantly inhibited the growth of LNCaP cells. MR42 was again the most effective and significantly better than MR2 .

Bispecific oligos are a significant advance in the design of antisense compounds and could play a role in treating prostatic cancer, particularly when they are administered with traditional chemotherapeutics.

Future Direction of Antisense Proposed

Antisense oligonucleotides (oligos) are linear strands of nucleotide bases synthesized in a sequence complementary to protein encoding mRNA. Translation arrest of the specifically targeted protein results, presumably as a result of oligo:mRNA hybrid degradation by RNAse H but may also be due to either DNA triplex formation first described in 1957 , protein binding interactions with growth factors, or other non-specific mechanisms which may not be dependent upon mRNA expression. This approach was initially directed against proteins associated with viral replication, but more recently has been applied to those which regulate cancer progression, such as growth factors, their receptors, apoptosis regulating protein bcl-2 or even translational factors. Most oligos are directed towards the encoding bases near or about the AUG initiation codon and until recently all administered oligos have been directed against single proteins. In order to increase biological action, several investigators have utilized single oligos having multiple mRNA binding activities. Some were single oligos which targeted several proteins based upon sequence homology, while one study included the simultaneous administration of two oligo compounds targeting unrelated (in base sequence) proteins. As described above, it was proposed that more than one type of specificity could be included along a single linear stretch of oligo bases. Bispecific oligos simultaneously target two mRNAs and theoretically enhance RNAse mediated degradation by forming a larger and more complex hybrid structure. Bispecifics have the additional advantages of directing with equal efficiency (since both active sites are expressed in tandem sequences on the same linear molecule) two different complementary binding sequences to the same targeted cell. They also have the ability to target mRNAs encoding proteins associated with different biochemical or growth regulatory pathways, as well as to simplify the delivery process.

Specific delivery of antisense oligos to targeted tissues is often a limitation of this technology. Other than an intra-ocular delivery system employed for the treatment of CMV retinitis, specific targeting of a systemically administered oligo is limited although progress has been made through innovative approaches which have been evaluated. A recent report describes a method where oligos were incorporated into microbubbles for systemic administration and then were specifically released in a targeted area using ultra low frequency sound waves. Indeed, the pathway by which antisense oligos enter cells to exert their effect is still uncertain. Mechanisms proposed have included receptor mediated transport and pinocytosis into endosomes. Those working with morpholino-type oligos derivatives insist that true exit out of endosomes require the (Gene Tools) Endo-Porter delivery system which promotes cytoplasmic entry and nuclear transport.

Other innovative models have been evaluated to enhance targeting and entry of oligos. These include the targeting of anti-oxidants to the mitochondria and also a method to induce apoptosis by directing a free radical spin trap into pancreatic cancer cells. Methods used to enhance delivery have also included conjugates with avidin, or poly-L-lysine acting as an electrostatic linker for targeting asialogylcoprotein receptors. Larger, polymetric complexes have also been evaluated which include the following components and functions: polymalic acid, which has multiple carboxyl groups to which conjugates can be attached (forming a multifunctional delivery system); morpholino-type oligos which are attached via a disulfide linkage, which is subsequently cleaved in the cytoplasm to release the drug; an antibody to the transferrin receptor which is attached to both facilitate localization to cancer cells and also to enhance receptor-mediated endocytosis; a short-chain polyethylene glycol (PEG) chain which is associated with L-leucine and L-valine to provide lipophilicity and to disrupt endosome membranes; lastly, a long-chain PEG is added for protection . Another method uses highly branched macromolecules with nanoscopic structures called dendrimeres. Protonated dendrimers alter the structure and function of negatively charged nucleic acids (oligos) by electrostatic interactions, while anime-terminated polypropylenimine dendrimers have been shown to condense antisense oligos and aid in the delivery process.

We propose solutions to several of the limitations associated with oligo development. First, branched oligos could be constructed which would include linear base sequences complementary to several (more than 2) mRNAs encoding different proteins. These oligos would be covalently bound through ester linkages (at intervals) to a hydrophobic lipid-like backbone carrier. For different tumor types the selection of oligos would be customized to target the particular proteins regulating that histologic type (or stage). Such a structure would have the advantage of multispecificity, equimolar delivery and enhanced membrane solubility. Secondly, this structure, with its hydrophobic regions, might be suitable to form liposomes, or to be incorporated into nanoparticle like microbubbles or dendrimeres. Release of branched oligos from either type of an enclosing structure could be accomplished by ultrasonic sound waves. The release of separate oligos from the branching structure for enhanced biological activity would be accomplished by intracellular esterases. Release from the carrier would eliminate any steric hindrance considerations.

2. Immuno- and histologic evaluation of tumor markers in Animal Studies

The effect of surgical and chemotherapy (paclitaxel) on tumor markers was evaluated in LNCaP tumors implanted into the flank of athymic nude mice. The initial study using immunohistochemistry evaluated tumor markers associated with growth, differentiation and angiogenesis. Another study in progress evaluates the effect of orchiectomy upon anti-angiogenic treatment (thalidomide) using both PC-3 and LNCaP models. Histologic markers to be evaluated are anaplasia, vascularity, mitotic index and apoptosis. Immunohistochemistry will include study of phosphorylated (activated) growth factor receptors. Additional treatment might also include antisense administration.

Abstract

Our aim is the identification and correlation of changes in tumor-associated protein expression which results from therapy. LNCaP tumors, excised from nude mice treated either by orchiectomy or with the chemotherapeutic agent paclitaxel, were evaluated for the expression of proteins and receptors associated with growth, differentiation and angiogenesis using immunohistologic procedures. Compared to untreated control tumors, both treatments reduced the expression of vascular endothelial growth factor (VEGF), prostate specific membrane antigen (PSMA), prostate specific antigen (PSA), androgen receptor (AR) and epidermal growth factor receptor (EGFR). The effect of paciltaxel treatment on AR expression was the most significant (p = 0.005). Of particular interest was identifying a significant correlation p = 0.000801) between PSMA and VEGF expression regardless of treatment modality. These altered expressions suggest that PSMA may also be a marker for angiogenesis and could represent a target for deliverable agents recognizing either prostatic tumors or endothelial development. Cell surface PSMA would then represent a unique target for treatment of patients early in their development of prostatic metastases.

3. Proliferative response of tumor cells under hypoxic or anoxic conditions

Abstract

Hypoxic cancer cells located beyond the diffusion path of sufficient oxygen are considered a nidus of therapeutic failure. Due to the dependence of many malignantly transformed cells on glycolysis for metabolic energy, inhibiting this and other sources of energy should seriously reduce cell viability and proliferation, additively or even synergistically. To try and duplicate in vitro some of the features of in vivo cancer cells likely to resist therapy, HeLa cells were incubated with sub-lethal concentrations of 2-deoxy-D-glucose under aerobic, hypoxic or virtually anoxic conditions, verified by increased synthesis of Hif-1α, and their replication and survival determined. MK 886, an inhibitor of mitochondrial function, was used to determine that organelle in energy metabolism. Culture of cervical cancer derived HeLa cells with 2-deoxy-D-glucose under these restrictive conditions did not reduce their proliferation or viability to the extent expected. Their surprisingly robust survival included the anticipated increase in dependence upon glycolysis and implied a likely entrainment of other constitutive and possible facultative energy sources and pathways. Increased synthesis of Hif-1α, increased binding to its consensus sequence and reduced inhibition from MK 886 in cells under oxygen deficient environments confirmed the presence of restrictive conditions. Efforts to suppress HeLa cell survival by reducing glucose consumption and metabolic energy from ambient oxygen may require inhibition of multiple energy sources, possibly not all of them identified. Assessment in vitro of agents directed against sources of metabolic energy or of other therapeutic agents against these or additional potential targets should include studies under hypoxia and relative anoxia. In this way, the likely response of in vivo hypoxic or anoxic cancer cells believed to contribute to therapeutic failure may be estimated.