PATHWAYS TARGETED BY THE OGF-OGFr AXIS ARE DETERMINANTS IN THE PROGRESSION OF HUMAN OVARIAN CANCER
Open Access
- Author:
- Donahue, Renee N.
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 01, 2011
- Committee Members:
- Patricia Mclaughlin, Dissertation Advisor/Co-Advisor
Patricia J Mc Laughlin, Committee Chair/Co-Chair
Ian Stuart Zagon, Committee Member
Ronald Paul Wilson, Committee Member
Robert Harold Bonneau, Committee Member
Michael Verderame, Committee Member - Keywords:
- Ovarian Cancer
Opioids
Opioid Growth Factor (OGF)
Opioid Growth Factor Receptor (OGFr)
Naltrexone (NTX)
Low Dose Naltrexone (LDN) - Abstract:
- Ovarian cancer is the leading cause of death from gynecological malignancies, and is the 5th leading cause of cancer related mortality among women. An estimated 225,000 new cases are diagnosed each year worldwide, resulting in 140,200 deaths annually. Approximately 90% of primary ovarian cancers are epithelial in origin, and the most common presentation (75%) is in the advanced stages (stage III/IV). Cytoreductive surgery and adjuvant chemotherapy serve as the major treatment modalities. Initial clinical response is excellent; however, nearly 65% of advanced-staged patients relapse within 2 years of initial therapy. Once ovarian cancer recurs, all subsequent treatments are palliative. The cellular and molecular events involved in ovarian cancer pathogenesis need to be defined, and major improvements in treatment will require new therapies based on exploitation of biological pathways. Dysregulation of cell proliferation is an integral part of the ovarian cancer phenotype. One native biological regulatory system of cell replication in normal cells and a variety of cancers is the opioid growth factor (OGF) and its receptor, OGFr. Chemically termed [Met5]-enkephalin, OGF is a constitutively active opioid peptide that is autocrine produced and secreted, and interacts with OGFr to delay the G1/S interface of the cell cycle by modulating cyclin-dependent kinase inhibitory (CKI) pathways, without affecting cell survival. The studies depicted in this thesis were aimed at establishing the role of the OGF-OGFr axis in the modulation of cell proliferation and determining the repercussions of modulating this axis on human ovarian cancer both in vitro and in vivo. The first study evaluated the presence, mechanism, and role of the OGF-OGFr axis on the modulation of the growth of OVCAR-3 and SKOV-3 human ovarian cancer cell lines. OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, serum-independent, reversible and receptor-mediated inhibitory action on cell proliferation that was dependent on RNA and protein synthesis. The repressive effect of OGF on cell proliferation also was also observed in CAOV-3 and HEY ovarian cancer cell lines. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr by siRNA technology stimulated cell replication, and abolished the inhibitory actions of exogenous OGF, documenting its integral role in mediating the effects of endogenous and exogenous OGF. The mechanism of OGF-OGFr action on DNA synthesis was related to the CKI pathways because knockdown of p16 and p21 in OVCAR-3 cells, and p21 in SKOV-3 cells eliminated OGF¡¦s inhibitory effect on growth. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in human ovarian cancer cells using an in vitro model system. The next study determined whether OGF inhibits tumor growth in mice with established subcutaneous xenografts of ovarian cancer, and determined whether the combination of OGF biotherapy with taxol or cisplatin chemotherapy (the standard of care) provides an additive inhibitory effect on ovarian cancer proliferation and tumor growth. Cell proliferation assays on SKOV-3 cells treated with OGF and/or taxol, or OGF and/or cisplatin, demonstrated that cell number and DNA synthesis were inhibited to a greater extent by combination treatments compared to individual treatments. The action of OGF, but not taxol or cisplatin, was mediated by a naloxone sensitive receptor, and was reversible and not toxic. Tumor volumes in mice with established subcutaneous xenografts were reduced up to 50% by OGF, up to 50% by taxol, and up to 58% by cisplatin treatment, compared to saline administered controls. Tumor volumes from the OGF plus taxol group were decreased 28% from mice treated with OGF alone, and 29% from mice treated taxol alone; tumor volumes in mice treated with OGF plus cisplatin were reduced 50% from mice treated with OGF alone, and 44% from mice treated with cisplatin alone. Importantly, OGF treatment in mice receiving cisplatin provided a significant degree of protection against the toxic effects of cisplatin (weight loss) seen in mice receiving cisplatin treatment alone. Evaluation of tumor tissue after 37 days of treatment revealed that taxol and cisplatin, but not OGF, induced apoptosis, while all treatments, markedly inhibited DNA synthesis and tumor angiogenesis; an additive inhibitory effect on DNA synthesis and angiogenesis was seen in mice receiving OGF with chemotherapies compared to mice receiving chemotherapy alone. Both OGF and OGFr were detected in tumor tissue, and OGFr binding and expression was reduced up to 51% and 81%, respectively, by OGF treatment, revealing a feedback mechanism. This preclinical evidence, demonstrates that OGF biotherapy markedly inhibits ovarian tumor growth in a non-toxic manner in vivo, and can be combined with taxol or cisplatin chemotherapy to provide an enhanced therapeutic benefit. We next investigated the repercussions of pharmacologically manipulating the OGF-OGFr axis in human ovarian cancer cells in vitro using the opioid receptor antagonist naltrexone (NTX), which is a nonselective opioid receptor antagonist that blocks the interaction of OGF and OGFr. It is well understood that the response to opioid antagonist administration is a compensatory upregulation in the production of endogenous opioids/opioid receptors. Pharmacokinetic and nociceptive studies have revealed that administration of a low dosage of NTX (LDN) in vivo, which blocks endogenous opioids from opioid receptors for a short period of time (4¡V6 h) each day, provides a window of 18¡V20 h for the upregulated opioids and opioid receptors to interact. In vivo daily administration of a low dosage of NTX (LDN, 0.1 mg/kg) has been shown to inhibit progression of colorectal cancer and neuroblastoma in xenograft models of cancer and is suggested to target cell proliferation. A tissue culture model of LDN, using short term NTX treatment, was established to investigate the mechanism of LDN in human ovarian cancer and to determine whether its action is independent of immune function and systemic factors. A single application of short term NTX (10-5 M, 6h) to SKOV-3 cultures inhibited cell number by 21-28% between 48 and 96 h. To determine which opioid peptide(s) mediate(s) the inhibitory actions of LDN on growth, cultures were exposed to various opioid peptides; under conditions and concentrations where OGF (10-6 M) inhibited cell number, none of the opioid peptides specific for the classical ƒÝ, ƒÔ, or ƒÛ opioid receptors altered growth. To establish whether the inhibitory actions of LDN are invoked by endogenous OGF, as well as determine the involvement of a particular opioid receptor, an OGF neutralizing antibody, and siRNA¡¦s against the ƒÝ, ƒÔ, ƒÛ, and OGF receptors were administered in combination with short term NTX; neutralizing OGF and silencing of OGFr, but not ƒÝ, ƒÔ, or ƒÛƒnopioid receptors, blocked the inhibitory action of LDN. An evaluation of the effects of short term NTX on the expression of OGF (RT-PCR, Immunohistochemistry, radioimmunoassay) and OGFr (Northern blot, Western blot, Immunohistochemistry, receptor binding) revealed a translational, but not transcriptional, upregulation of both peptide and receptor. To ascertain whether LDN¡¦s inhibitory action is related to decreased survival and/or proliferation, TUNEL, trypan blue staining, and BrdU incorporation were evaluated. Beginning 12 h and persisting 66 h after a single application of short term NTX, DNA synthesis was decreased up to 48%, with no alterations in cell survival noted. Silencing of p16 and/or p21 blocked the inhibitory action of short term NTX indicating a mechanism directed to the CKI pathways. This study demonstrated that short term opioid receptor antagonism, the equivalent of LDN, inhibited ovarian cancer cell proliferation independent of immune function and systemic factors, through upregulation of the OGF-OGFr axis. Upregulation of the OGF-OGFr axis by short term opioid receptor blockade with LDN may provide a novel target for biotherapy of ovarian cancer. The next study evaluated whether LDN inhibits tumor growth in mice with established subcutaneous xenografts of ovarian cancer, and determined whether the combination of LDN biotherapy with taxol or cisplatin chemotherapy (the standard of care) provides an additive inhibitory effect on ovarian cancer proliferation and tumor growth. Cell proliferation assays on SKOV-3 cells treated with LDN and/or taxol, or LDN and/or cisplatin, demonstrated that cell number and DNA synthesis rates were inhibited to a greater extent by combination treatments compared to individual treatments. The action of LDN, but not taxol or cisplatin, was reversible and not toxic. Tumor volumes in mice with established subcutaneous xenografts were reduced up to 48% by LDN, up to 54% by taxol, and up to 54% by cisplatin treatment, compared to saline administered controls. Tumor volumes from the LDN plus taxol group, were comparable to those in mice treated with LDN or taxol alone; however, tumor volumes in mice treated with LDN plus cisplatin were reduced 35% from mice treated with LDN alone, and 37% from mice treated with cisplatin alone. Importantly, LDN treatment in mice receiving cisplatin provided a significant degree of protection against the toxic effects of cisplatin (weight loss) seen in mice receiving cisplatin treatment alone. Evaluation of tumor tissue after 35 days of treatment revealed that taxol and cisplatin, but not LDN, induced apoptosis, while all treatments, markedly inhibited DNA synthesis and angiogenesis; an additive inhibitory effect on DNA synthesis and angiogenesis was seen in mice receiving LDN with cisplatin, but not LDN with taxol, compared to mice receiving chemotherapy alone. Both OGF and OGFr were detected in tumor tissue. Revealing the mechanism and compensatory upregulation of the OGF-OGFr axis, OGF expression was increased 37% by LDN treatment, and OGFr binding and expression was increased up to 133% and 46%, respectively, by LDN treatment, compared to saline administered controls. This preclinical evidence, demonstrates that LDN biotherapy markedly inhibits ovarian tumor growth in a non-toxic manner in vivo, and can be combined with cisplatin, but not taxol chemotherapy to provide an enhanced therapeutic benefit. We next investigated the effects of OGF or LDN on ovarian cancer tumor growth using what is considered to be a more clinically relevant model of human ovarian cancer, where cancer cells are injected and grown in the intraperitoneal cavity as opposed to subcutaneously. Immediately after tumor cell inoculation, mice were treated with OGF, LDN, or an equivalent volume of saline. At the end of 40 days of treatment, the number of tumor nodules was decreased 41% and 38% by OGF and LDN, respectively, and tumor mass was reduced 68% and 46%, by OGF and LDN, respectively, compared to saline administered controls. This preclinical evidence, using the more clinically relevant ovarian cancer model, suggests that OGF or LDN biotherapy markedly inhibits ovarian tumor growth by inhibiting tumor cell proliferation and angiogenesis, without inducing apoptosis. Molecular regulation of tumor cell progression was studied in two independent experiments whereby constructs to overexpress OGFr or underexpress OGFr were established and characterized both in culture and subsequently in nude mice. Using SKOV-3 cells, 5 clonal lines overexpressing OGFr were examined and demonstrated increases in OGFr protein expression, as measured by semiquantitative immunohistochemistry and Western immunoblotting. OGFr binding assays of clonal lines revealed 51-154% increases in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. Under standard growth conditions, cell number in clonal lines overexpressing OGFr was decreased by 31-85%, and doubling times were extended 41-177% compared to WT and EV cultures. Nude mice injected subcutaneously or intraperitoneally with clonal cell lines overexpressing OGFr had increased latencies to tumor formation, and the tumors were markedly decreased in volume/number, weight, and exhibited increases in OGFr expression compared to mice injected with WT or EV cells. These data on the stable overexpression of OGFr indicate that OGFr is vital to cell replicative events in human ovarian cancer both in vitro and in vivo, and support treatments that amplify OGFr to decrease the growth of these neoplasias. shRNA constructs were prepared to knockdown OGFr in SKOV-3 cells; 2 clonal lines were examined. OGFr protein expression was decreased up to 73% in clones compared to wild-type (WT) and empty vector (EV) controls; binding assays of clones revealed 50-55% decreases in binding capacity compared to controls. Growth and DNA synthesis were increased 33-146%, and doubling times decreased 29-35% compared to WT and EV cultures. Nude mice injected subcutaneously with cells underexpressing OGFr had an increased tumor incidence, decreased latency to tumor formation, and tumors were increased in volume and decreased in OGFr expression compared to WT and EV controls. OGF treatment in mice with WT or EV tumors, but not OGFr underexpressing tumors, markedly inhibited tumor volume, weight, and DNA synthesis. These data on the stable underexpression of OGFr demonstrate the critical nature of the OGF-OGFr axis in the progression of human ovarian cancer. In summary, upregulation of the OGF-OGFr axis by treatment with exogenous OGF, pharmacologic upregulation of OGF and OGFr by short term opioid receptor antagonism with LDN, or stable molecular overexpression of OGFr, all inhibited ovarian cancer cell proliferation and tumor growth in a non-toxic manner. This pre-clinical data supports treatment modalities that amplify OGF/OGFr to decrease the growth of these neoplasias. The critical role of OGFr in the progression of this cancer was documented through experiments evaluating the repercussions of stably underexpressing OGFr, and will be important in understanding factors determining the success/failure of therapeutic modalities.