Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

Immune response to regressor and progressor tumor variants

Most skin cancers induced in mice by Ultraviolet (UV) radiation express highly immunogenic Tumor specific transplantation antigens (TSTAs) and thus exhibit a regressor phenotype. In this study, I have used cloned genes encoding tumor antigens and oncogenes in conjunction with DNA transfection technique to isolate and characterize regressor variants from progressor tumors and vice versa. The purpose of this study was (1) to determine whether the product of a cloned gene (216) from UV-1591 tumor, which encodes a novel MHC class I antigen can function as a tumor rejection antigen when expressed on unrelated, nonantigenic, murine tumor cells or whether its function is restricted to UV-induced tumors, and (2) to determine the processes by which progressor variants derived from a regressor UV-2240 cell line by transfection with an activated Ha-ras oncogene escape the immune defenses of the normal immunocompetent host.^ To answer the first question, a spontaneously transformed, nonimmunogenic cell line (10T-1) was cotransfected with DNA from p216 and pSV2-neo plasmids. Results demonstrate that the product of a cloned TSTA gene from a UV-induced murine tumor is capable of functioning as a tumor rejection antigen when expressed on unrelated, nonantigenic tumor cells. In addition, these results indicate that this approach could be used to augment the immune response against poorly antigenic tumors.^ To answer the second question, progressor variants were isolated from a highly antigenic UV radiation-induced C3H mouse regressor fibrosarcoma cell line, UV-2240, by transfection with an activated Ha-ras oncogene. Subcutaneous injection of Ha-ras-transfected UV-2240 cells into immunocompetent C3H mice produced tumors in 4 of 36 animals. In addition, the Ha-ras-induced progressor variants produced experimental lung metastasis in both normal C3H and nude mice, although they induced more lung nodules in nude mice than in normal C3H mice. Results indicate that the progressor phenotype of the Ha-ras-induced tumor variants is not due to loss of TSTAs or MHC class I antigens. This implies that some tumors can escape the immune defenses of the normal immunocompetent host by mechanisms other than the loss of TSTAs and MHC class I antigens. (Abstract shortened with permission of author.) ^

The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

The role of nitric oxide in apoptosis and cancer metastasis

The purpose of these studies was to investigate the role of nitric oxide (NO) in tumor metastasis. K-1735 Metastatic cells survived in blood circulation to produce experimental lung metastases, whereas nonmetastatic cells did not. After incubation with combination cytokines or lipopolysaccharide (LPS), nonmetastatic cells exhibited high levels of inducible nitric oxide synthase (iNOS) activity and NO production, whereas metastatic cells did not. The production of NO directly correlated with cytotoxic effects of cytokines or LPS. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases, highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.^ Furthermore, multiple systemic administrations of multilamellar vesicle-liposomes (MLV) containing the lipopeptide CGP 31362 (MLV-31362) or MLV-31362 combined with murine interferon-gamma (IFN-$\gamma$) eradicated the metastases by M5076 reticular cell sarcoma. Tumor regression correlated with iNOS expression within the tumor lesions and with increased NO production. The administration of NMA significantly decreased NO production and diminished the antitumor activities. These data imply that the activation of iNOS can serve as a target for immunotherapeutic agents for treatment of murine reticulum cell sarcoma metastases. ^

Radiolabeled human monoclonal IgM for intracompartmental cancer therapy

Radioimmunotherapy (RIT) with i.v. administered radiolabeled IgG can selectively irradiate tumor cells in vivo. However, it only provides effective therapy for lymphomas. Intracompartmental RIT with radiolabeled human monoclonal IgM may allow curative treatment of solid tumors by increasing tumor deposition of radioactivity, reducing systemic toxicity and allowing repeated administration. This hypothesis was tested in nude mouse models with IgM radiolabeled with indium-111 $\rm(\sp{111}In)$ or yttrium-90 $\rm(\sp{90}Y).$ The use of two radioisotopes, $\rm\sp{111}In$ for imaging and $\rm\sp{90}Y$ for therapy, allow for more quantitative and cautious development of RIT.^ Radiolabled 2B12, an IgM reactive with human ovarian carcinomas was tested by i.v. and intraperitoneal (i.p.) administration in nude mice bearing i.p. nodules of a human ovarian carcinoma cell line (SKOV3 NMP2). Radiolabeled CR4E8, an IgM reactive with human squamous cell carcinomas was tested by i.v. and intralesional (i.l.) administration in nude mice bearing subcutaneous tumors of a human head and neck squamous cell carcinoma cell line (886). These two models were selected to test proof of concept. Radiolabeled irrelevant IgM (CH-1B9), and $\rm\sp{90}Y$-aggregate served as specificity controls. Biodistribution was performed by excising, weighing and then measuring the radioactivity of tumor and normal organs. Therapy was conducted with i.p. $\rm\sp{90}Y$-labeled 2B12 using both single and fractionated administration and with i.l. $\rm\sp{90}Y$-labeled CR4E8 using single administration. Mice were monitored for tumor response, survival and systemic toxicity.^ Intracompartmental administration of radiolabeled IgM produced immediate high and prolonged tumor deposition of radioactivity with low normal tissue uptake. In contrast, i.v. administration resulted in low tumor, but high liver and spleen uptake. Similar biodistributions were demonstrated for $\rm\sp{111}In$- and $\rm\sp{90}Y$-labeled IgM. Intraperitoneal therapy with $\rm\sp{90}Y$-labeled 2B12 increased survival by approximately 12 days for every 100 $\rm\mu Ci$ of activity without significant toxicity for single (0-300 $\rm\mu Ci)$ and fractionated (150-510 $\rm\mu Ci)$ administration. Intralesional therapy with $\rm\sp{90}Y$-labeled CR4E8 (150-400 $\rm\mu Ci)$ induced prolonged complete regressions. Significant local or systemic toxicity was not observed.^ Intracompartmental RIT with radiolabeled tumor-reactive human monoclonal IgM can selectively irradiate tumor cells. Intracompartmental radiolabled IgM can significantly extend the survival of treated mice with minimal toxicity. It deserves further development as a new cancer therapy. ^

Development of prostate cancer gene therapy

Osseous metastases account for most of the morbidity and mortality associated with prostate cancer, for which there are currently no effective therapies. In the skeletal metastatic environment, neoplastic prostatic epithelial cells interact in a bidirectional stimulatory manner with osteoblastic stromal cells. Similarly, the presence of osteoblastic cells is essential for the survival and maintenance of intraosseous prostate cancer cells. In this thesis, I have developed novel gene therapy strategies for the treatment of androgen-independent human prostate cancers in experimental animal models. First, Ad-CMV-p53, a recombinant adenovirus (Ad) containing p53 tumor suppressor gene driven by the universal cytomegalovirus promoter, was effective in inhibiting prostate cancer cell growth, and direct intratumoral injections of Ad-CMV-p53 resulted in tumor regression. Second, because prostate cancer cells as well as osteoblastic cells produce osteocalcin (OC), OC promoter mediated tissue/tumor specific toxic gene therapy is developed to interrupt stromal-epithelial communications by targeting both cell types. Ad-OC-TK, a recombinant Ad containing the herpes simplex virus thymidine kinase (TK) gene driven by the OC promoter, was generated to inhibit the growth of osteoblastic osteosarcoma with prodrug acyclovir (ACV). Ad-OC-TK/ACV also inhibited the growth of prostate cancer cells and suppressed the growth of subcutaneous and intraosseous prostate tumor. In order to combine treatment modalities to maximize tumor cell-kill with minimized host toxicities, Ad-OC-TK/ACV was applied in combination with low dose methotrexate to eradicate osteoblastic osteosarcoma. In targeting of micrometastatic disease, intravenous Ad-OC-TK/ACV treatment resulted in significant tumor nodule reduction and prolonged the survival of animals harboring osteosarcoma lung metastases without significant host toxicity. Ad-OC-TK is a rational choice for the treatment of prostate cancer skeletal metastasis because OC is uniformly detected in both primary and metastatic human prostate cancer specimens by immunohistochemistry. Ad-OC-TK/ACV inhibits the growth not only of prostate cancer cells but also of their supporting bone stromal cells. Targeting both prostate cancer epithelium and its supporting stroma may be most efficacious for the treatment of prostate cancer osseous metastases. ^

Developmental effects of estrogen and PCBs in the reproductive tract of BALB/C mice

Many of the tumorigenic effects that result from neonatal exposure to both natural and synthetic estrogens resemble those found in humans exposed to diethylstilbestrol (DES) in utero. Using this established DES neonatal mouse model, my goal was to investigate long-term molecular and morphological effects of certain polychlorinated biphenyls (PCBs) that are weakly estrogenic in adult mice. Focusing on the cervicovaginal (CV) tract, since this is where tumors develop in the BALB/c mouse, I first assessed the 17β-estradiol (E2) dose-response for expression of lactoferrin (LTF). LTF is a highly inducible estrogen biomarker that is permanently altered in uteri from neonatally treated mice. Treatments were administered via 5 subcutaneous injections beginning within 16 hrs after birth, days 1–5. ^ The ontogeny of LTF expression from mouse CV tracts was determined by examining three different stages of life: pups, immature, and mature mice. Northern RNA analysis and immunohistochemistry showed that neonatal E 2 treatment both increases and decreases LTF expression. Early expression of LTF in the CV tract at all doses occurred in pups. In both immature and adult mice, increased LTF expression was dependent on whether E2 induced ovary-dependent or ovary-independent persistent vaginal cornification. ^ Next, I studied biological responses from neonatally PCB exposed adult mice. As expected, using a neonatal uterine bioassay I showed that 2 ′4′6′-trichloro-4-biphenylol (OH-PCB-30), 2′3′4′ 5-tetrachloro-4-biphenyloI (OH-PCB-61), and OH-PCB-30/61 (50/50 mixture), were estrogenic causing a dose-dependent increase in uterine weight. ^ Long-term effects of OH-PCB 30 [200 μg/pup/day] were most similar to E2 as seen by an increased uterine wet weight in day 50 mice similar to E2 [5 μg/pup/day] (141% and 140% of control, respectively). Another similarity between OH-PCB 30 and E2 neonatally treated mice was found in those sacrificed at 20 months of age. At these same doses CV tract squamous cell carcinoma induction was 43% of E2 treated mice and 47% of OH-PCB 30 treated mice. Differences were noted in adenoaquamous; cell carcinoma development, where 16% of OH-PCB-30 neonatally treated mice developed tumors versus 8% for E2. Based on these results using the neonatal mouse model, I conclude that the OH-PCBs tested are strongly estrogenic and tumorigenic showing dose-response relationships when exposure occurs during development of the reproductive tract in mice. These results may have important implications for risk assessment in determining the effects of xenoestrogens exposure early versus later in life. ^