Progesterone Induced Blocking Factor Protein Research Articles

Abstract 3587: Evidence that membrane progesterone receptors (mPR) play a key role in cancer metastases in most cases of metastatic cancer

Abstract An immunomodulatory protein called the progesterone induced blocking factor (PIBF) is essential for the fetal semi-allograft to escape immune surveillance. Many cancer cell lines make both mPRs for PIBF and the protein itself. The progesterone receptor (PR) antagonist mifepristone added to cancer cell line cultures down-regulated mRNA for PIBF and the PIBF protein. This IRB approved study (that required a compassionate use IND from the FDA) evaluated oral mifepristone 200mg per day (majority) or 300mg in patients with very advanced cancers with no more treatment options available. Patients would remain under the care of their primary oncologists (FDA requirement). Because the nuclear (n) PR may be protective against cancer spread, only patients with cancers negative for the nPR were treated. Patients with the following types of cancers received mifepristone: non-small cell lung cancer (NSCLC) (n=9), small cell lung cancer (SCLC) (n=2), colon (n=3), pancreatic (n=2), thymic epithelial cell (n=1), transitional cell carcinoma of the renal pelvis (n=1), malignant fibrous histiocytoma (n=1), glioblastoma multiforme stage IV (n=1), leiomyosarcoma (n=1). All 21 patients showed significant palliative benefits, especially but not limited to, marked reduction in pain and marked improvement in energy, decreased dyspnea on exertion, and return of mental clarity. For those who had repeat CT evaluation no one failed to show shrinkage and decrease in malignant lesions and 2 (leiomyosarcoma and SCLC) showed complete remission after a few months on mifepristone. Five patients (3 females, 2 males) with SCLC (1), NSCL (n=3) and fibrous osteosarcoma (n=1) survived at least 5 years with 2 dying from cancer unrelated causes (SCLC and NSCLC). Two are still alive (NSCLC with EGFR mutation and brain metastases). The male with fibrous osteosarcoma died after stopping mifepristone in favor of a new drug recommended by his oncologist. Two patients lived over 2 years. Most would have lived longer had they not stopped mifepristone. Other observations: significant palliative benefits are seen within 3 weeks of taking mifepristone before any evidence of significant tumor regression. After a period of time, some lesions (especially in the primary location) may start growing slowly, but this is not a sign of loss of drug efficacy and long good quality life is still probable if one remains on the drug. The cancer spreads rapidly if one stops the mifepristone. The usual reason for stopping the drug was intervention by the primary oncologist suggesting a new clinical trial because there has not been complete remission of the cancer or some tumor growth (usually slow) or finances. Not one patient had a side effect. Hopefully this study will generate interest in treating patients with advanced cancer with mifepristone, especially those devoid of the nPR. These data support the role of mPRs in cancer proliferation. Citation Format: Jerome H. Check, Diane Check, Brooke Neumann, Carrie Wilson. Evidence that membrane progesterone receptors (mPR) play a key role in cancer metastases in most cases of metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3587.

Abstract 605: Evidence that a wide variety of cancers may use the progesterone induced blocking factor protein to escape immune surveillance as evidenced by significant palliation following treatment with progesterone receptor antagonists in advanced metastatic treatment resistant cancers

Abstract A search of the literature and abstracts from national oncology meetings was conducted to determine what types of human cancers have been reported to show some type of palliative benefit from treatment with progesterone (P) receptor antagonists. Though the original concept was to try P receptor antagonists for cancers known to be associated with the classic nuclear P receptor, recent data suggest that the real beneficial target is a membrane P receptor that helps produce a unique immunomodulatory protein called the progesterone induced blocking factor (PIBF) that inhibits natural killer (NK) cells and cytotoxic T cells in the tumor microenvironment and thus may allow cancer cells to metastasize. The following human cancers known to be associated with the classic nuclear receptor that have responded to either the P receptor modulator mifepristone or onapristone, include breast, uterine, ovarian, and prostate cancer. Human cancers not associated with the classic nuclear P receptor that have demonstrated significant palliative benefits and/or increased longevity following P receptor modulator therapy include colon, thymic cell epithelial cancer, transitional cell carcinoma of the renal pelvis, leiomyosarcoma, pancreatic carcinoma, malignant fibrous histiocytoma, glioblastoma multiforme grade IV, renal cell carcinoma, fibrous osteosarcoma, small cell lung cancer, non-small cell lung cancer (including those negative for tumor markers, or those positive for PD-1, or positive for the EGFR mutation). The wide variety of very advanced cancers that have responded to the P receptor modulators as evidenced by extension of a better quality of life despite the fact that they had progressed despite standard chemo or immunotherapy, is consistent with the concept that PIBF may be a protein needed by a large variety of cancers to advance or metastasize, and thus may provide a unique target to treat with P receptor modulators, and thus provide significant palliative benefit. In contrast to most anti-cancer agents, P receptor antagonists are extremely well tolerated, and are without serious risk of complications. Larger series are needed to determine the true beneficial effect of this type of therapy. Positive beneficial effects in case reports, at least provide the basis for selection of which cancers to evaluate in a larger series. Citation Format: Jerome H. Check, Diane Check, Carrie Wilson. Evidence that a wide variety of cancers may use the progesterone induced blocking factor protein to escape immune surveillance as evidenced by significant palliation following treatment with progesterone receptor antagonists in advanced metastatic treatment resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 605.

Abstract 695: Treatment with oral mifepristone enables a patient with end-stage pancreatic cancer in hospice, on a morphine drip, to restore a decent quality of life

Abstract The progesterone (P) receptor modulator, mifepristone, has been previously found to provide significant improvement in length and quality of life in people with a variety of advanced cancers. A 57 year old man with severe abdominal pain, dyspnea, nausea weakness and vomiting, in hospice with marked sedation from a morphine drip, was treated with oral mifepristone 200mg/day, as an option to extend his lifespan with reasonable quality of life. He had never received treatment for his disease and was given a prognosis of only 1-3 weeks to live, by his oncologist. He showed quick improvement in both his nausea (which was intractable), and his abdominal pain, within one month of treatment. His first visit he was wheelchair ridden, next visit he had progressed to a cane and after 2 months walked in without any assistance. He has now been on oral mifepristone 200mg/day for 5 months, as his only treatment. His only complaint is slight weakness and mild dyspnea; he is considered ECOG-2 when initially he was ECOG-IV. He has had no side effects from the mifepristone and rarely needs his previous analgesics, or anti-emetics. The hypothesized mechanism of action of this drug is to inhibit the activation of a membrane P receptor in the cancer cells that causes the production of a 757 amino acid unique protein measuring 90 kDa that is involved in cell-cell regulation, but is converted to smaller intracytoplasmic splice variants, which are immunosuppressive proteins. It is called the progesterone induced blocking factor (PIBF). When these smaller splice variants are released into the tumor micro-environment, they suppress cytolytic activity of the cellular immune system, e.g., by stabilizing perforin granules in natural killer cells. The fact that treatment with mifepristone has been shown to improve length and quality of life, not only in cancer associated with the classic nuclear P receptor, e.g., breast, ovarian, and endometrial cancer, but also those cancers devoid of the classic nuclear receptor, e.g., small cell and non-small cell lung cancer, leukemia, colon cancer, thymic epithelial cell cancer, renal cell carcinoma, transitional cell carcinoma of the renal-pelvis, and glioblastoma multiforme, suggests that the PIBF protein may be important for a large variety of cancers to escape immune surveillance. Pancreatic cancer can be added to this list of advanced cancers that respond to mifepristone therapy. The fact that it seems to work even when standard chemotherapy, immunotherapy, and targeted therapies have failed to work on patients with widespread cancer, including a large variety of cancers, could suggest that the PIBF protein may be a mechanism used by stem cells to escape immune surveillance. Since there are no other treatment options, we have elected not to repeat computerized tomography scans to evaluate whether the lesions have regressed but we believe clinically they must have. Citation Format: Jerome H. Check, Diane Check, Carrie Wilson, Maya Srivastava, Trina Poretta, James Aikins. Treatment with oral mifepristone enables a patient with end-stage pancreatic cancer in hospice, on a morphine drip, to restore a decent quality of life [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 695.

Abstract 696: Palliative benefits of oral mifepristone treatment for metastatic fibroblastic osteosarcoma

Abstract Treatment with the progesterone receptor modulator, mifepristone, has provided patients with advanced adenocarcinomas, even those devoid of the classic nuclear progesterone (P) receptor, who had previously progressed off of standard chemotherapy, immunotherapy, or targeted therapy, significant palliative benefits and extension of life. The purpose of this research was to determine if mifepristone could similarly provide benefits for a sarcoma, namely, a metastatic fibroblastic osteosarcoma, that had progressed despite treatment with surgery, radiation, and chemotherapy. A 51 year old male was diagnosed at age 46, with an osteosarcoma of the right leg. He had neoadjuvant radiation therapy and a resection of the right proximal tibia to remove a 6cm tumor. This was followed by a chemotherapy cocktail of doxorubicin, cisplatin and high dosages of methotrexate for 9 months. Unfortunately, a tumor recurred and it was again resected. Since he was also found to have a 1.6 × 1.3cm upper lung nodule and another right lower lung nodule, he was also treated with ifosamide and etoposide for 5 months. Subsequently, these 2 drugs were continued for another 6 months alternating with high dose methotrexate. Foundation one testing revealed targeted mutations for CD104, FGRI, and KRAS, leading to treatment with a targeted therapy, regorafenib. However, 8 months later imaging studies showed progression of disease. Mifepristone, 200mg orally, was then added. CT scans had shown a steady decline in his condition until mifepristone was started. After 3 months of therapy, he showed stable disease with no increase in size of his metastatic lesions, or any new ones. He feels better on mifepristone than he has felt on any of his previous therapies, even when his disease was not as advanced. He has now completed five months of the combination therapy. He has neuropathies, manifested as pain in his hands and feet and somnolence from the regorafenib and no new side effects from mifepristone. This is the first non-gynecologic sarcoma, not known to be associated with the presence of the nuclear P receptor, reported to respond to mifepristone. There had been a previous case of widely metastatic leiomyosarcoma that showed dramatic response to mifepristone. The hypothesized mechanism of action for mifepristone in inhibiting growth and spread of metastatic cancer, is by inhibiting the production of the immunomodulatory protein the progesterone induced blocking factor (PIBF), actually secreted by the cancer cells themselves into the tumor microenvironment that results from activation of membrane P receptors. PIBF protein suppresses the cellular immune reaction to the tumor cells. Mifepristone, by blocking this membrane P receptor, inhibits PIBF production. Fibroblastic osteosarcoma can be added to the long list of advanced cancers, resistant to standard cancer therapy, yet demonstrating palliative benefits from mifepristone treatment. Citation Format: Jerome H. Check, Diane Check, Trina Poretta, James Aikins, Carrie Wilson. Palliative benefits of oral mifepristone treatment for metastatic fibroblastic osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 696.

The immunolocalization of Galectin-1 and Progesterone-Induced Blocking Factor (PIBF) in equine trophoblast: Possible roles in trophoblast invasion and the immunological protection of pregnancy

IntroductionThe proteins galectin-1 and Progesterone Induced Blocking Factor (PIBF) are present on human and murine trophoblast and are thought to influence both immunomodulation and trophoblast invasion. In equids, the invasive component of the placenta, the endometrial cups, stimulate maternal cell-mediated and humoral immune responses. It was therefore of interest to know if galectin-1 or PIBF could be immunolocalised to the invasive and/or non-invasive components of the equine placenta. MaterialsHorse and mule (♀ horse X ♂ donkey) embryos and placental tissues between Days 12 and 124 of gestation were stained immunohistochemically with antibodies raised against galectin-1 and PIBF. Results: Galectin-1 stained the non-invasive trophoblast between Days 15 and 20 but thereafter stained only the invasive trophoblast cells of the chorionic girdle, both before and after they invaded the endometrium to form the endometrial cups. PIBF, on the other hand, stained both the invasive and non-invasive trophoblast throughout the period of gestation studied. Of particular interest was the relative lack of staining of the endometrial cup cells in mule compared to horse pregnancies for galectin-1 and PIBF prior to the earlier and more rapid death and desquamation of the mule cup cells. Discussion: The expression of galectin-1 and PIBF proteins in equine trophoblast and the marked difference in lifespan between the endometrial cups in intraspecies horse versus interspecies mule pregnancies support a likely role for these two proteins protecting the fetal trophoblast from maternal immune attack and/or modulation of the invasiveness of endometrial cup cells.

Pregnancy-associated changes in expression of progesterone receptor and progesterone-induced blocking factor genes in bone marrow of ewes

Progesterone (P4) regulates reproductive and immune functions through binding to the progesterone receptor (PGR), and the effects of P4 are partly mediated by a progesterone-induced blocking factor (PIBF). Bone marrow (BM) is a key component of the lymphatic system and has an important role in immune response. In this study, BM was harvested from femurs on days 13, 16 and 25 of pregnancy and day 16 of the estrous cycles without mated by intact rams, and a qRT-PCR assay, Western blot and an immunohistochemistry analysis were used to analyze the expression of PGR and PIBF genes in BM. The results showed that there was an increase in relative abundance of PGR and PIBF mRNA in BM during early pregnancy, and PGR-B and the full-length PIBF genes were up-regulated in pregnant ewes. Immunohistochemistry results confirmed that the PGR and PIBF proteins were localized in both the cytoplasm and nuclei of adipocytes and the cells in the stroma and capillaries. This is the first study reporting an up-regulated expression of PGR-B and full-length PIBF genes in BM during early pregnancy in sheep. It is suggested that the conceptus exerted its effects on the adipocytes and the cells in the stroma and capillaries in BM, which were involved in the immunoregulation of BM through both cytosolic and nuclear pathways in ewes.

Effect of early pregnancy on the expression of progesterone receptor and progesterone-induced blocking factor in ovine lymph node

Lymph nodes are the sites where the immune reaction or suppression takes place. Progesterone (P4) exerts an essential effect of the immunomodulation on the maternal uterus during early pregnancy in ruminants. At present study, the inguinal lymph nodes were obtained at day 16 of non-pregnancy, days 13, 16 and 25 of pregnancy (n = 3 for each group) in ewes, and RT-PCR assay, western blot and immunohistochemistry analysis were used to analyze to the effect of early pregnancy on the expression of P4 receptor (PGR) and progesterone-induced blocking factor (PIBF) in the lymph nodes. Our results showed that the PGR and PIBF mRNA were up-regulated in the lymph nodes in pregnant ewes, and the PGR isoform (60 kDa) and the PIBF variant (75 kDa) were expressed constantly in the lymph nodes. However, there was no expression of the PGR isoform (40 kDa) and the PIBF variant (48 kDa) at day 16 of the estrous cycle. The immunohistochemistry results confirmed that the PGR and PIBF proteins were limited to the subcapsular sinus and trabeculae in the cortex, medullary sinuses, and were localized in the cytoplasm of the specific cells. This paper reports for the first time that early pregnancy exerts its effect on the specific cells in the lymph nodes through P4, which results in the up-regulated expression of the PGR mRNA and 40 kDa isoform, the PIBF mRNA and 48 kDa variant, and is involved in the immunoregulation of the lymph nodes through a cytosolic pathway in ewes.

Abstract 4096: A pilot study was initiated to determine if the immunomodulatory protein, the progesterone-induced blocking factor (PIBF), is present in higher quantity in the sera of patients with gynecologic cancer as compared to controls without cancer

Abstract PIBF is a unique protein that is secreted by gamma/delta T cells and precipitously rises in the sera following progesterone (P) exposure. The 34 kDa protein protects the fetal semi-allograft from immune surveillance by reducing cytoxicity of natural killer (NK) cells by stabilizing perforin granules. The parent 90 kDa form occupies a centrosomal position close to BRCA-1. PIBF can be found in most rapidly dividing cells. One study found an over-expression of the mRNA for the PIBF protein in all 29 leukemia cell lines tested. The P receptor antagonist mifepristone was found to down-regulate PIBF protein. Mifepristone provided significant palliative benefit to a large variety of spontaneous murine and human cancers. P could also be responsible for converting the 90 kDa parent form to a 34-36 kDa split variant in the cytoplasm which may be the immunosuppressive form in cancer cells. Alternatively, some P-like secretion by the tumor could influence gamma/delta T cells in the tumor microenvironment to secrete PIBF and possibly spill over to the sera. The aim of this study was to determine if there is any increase in serum PIBF in women with gynecologic cancer as opposed to controls. Serum was obtained from women about to have surgery for gynecologic problems including malignant and benign disorders. The samples would then be measured for PIBF using a new non-commercial enzyme linked immunoabsorbent assay (ELISA) for PIBF and serum progesterone. The PIBF levels (ng/mL) from lowest to highest in women with various gynecologic cancers (in all cases serum progesterone (P) <2ng/mL) were 10.06, 17.35, 32.59, 35.62, 54,7, and 57.17. The average serum PIBF was 34.6 ng/mL. There were 3 women with benign gynecologic tumors and the serum PIBF was 14.76, 15.7, and 36.64 their average serum PIBF was 22.5 ng/mL). There were 2 women with no tumors having gynecologic surgery and their serum PIBF levels (ng/mL) were 9.56 and 35.27 (with an average of 22.4 ng/mL). At least for gynecologic cancers if PIBF is conferring immune protection it is more likely operating through its intracytoplasmic presence rather than working through sera levels. In women or men exposed to exogenous or endogenous P for just 6 days it is not unusual to see sera PIBF rise to 300 to >800ng/mL. Citation Format: Jerome H. Check, Mojirayo Sarumi, Ann DiAntonio, Krystal Hunter, Gunda Simpkins, Marie Duroseau. A pilot study was initiated to determine if the immunomodulatory protein, the progesterone-induced blocking factor (PIBF), is present in higher quantity in the sera of patients with gynecologic cancer as compared to controls without cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4096. doi:10.1158/1538-7445.AM2014-4096

Progesterone-induced blocking factor is hormonally regulated in human astrocytoma cells, and increases their growth through the IL-4R/JAK1/STAT6 pathway

Astrocytomas are the most frequent and aggressive primary brain tumors in humans and constitute the leading cause of brain cancer related deaths. There are reports indicating that progesterone (P4) participates in the growth of astrocytomas through the interaction with its intracellular receptor (PR). Recently, it has been found that P4 induces the growth of several tumors through the up-regulation of progesterone-induced blocking factor (PIBF), a protein that has been related to the immunologic and proliferative actions of P4. U373 cells derived from a human astrocytoma grade III were used to study the role of P4 in PIBF expression and the effects of the latter in cell number. By using RT-PCR and Western blot techniques, we found that U373 cells express PIBF mRNA and protein. P4 (10nM and 100nM) increased PIBF mRNA expression after 1 and 3h of treatment, respectively, and this increase lasted 24h. This effect was blocked by the PR antagonist, RU486. Two PIBF isoforms were detected: one of 57kDa and the predominant one of 90kDa. The content of the 90kDa isoform increased after 12h of P4 treatment, and RU486 also blocked this increase. We observed that PIBF was released into the extracellular medium, being the 57kDa isoform the most abundant in this compartment. Immunofluorescence analysis showed that PIBF was localized in both the cytoplasm and nucleus. The effects of PIBF on cell number were analyzed for five consecutive days. PIBF (200ng/mL) significantly increased the number of U373 cells on days 2–5. Co-immunoprecipitation and Western blot assays revealed that PIBF associates to IL-4 receptor, and increases JAK1 and STAT6 phosphorylation at 20min. Our results suggest that P4 regulates PIBF expression in U373 cells through PR, and that PIBF increases cell number through IL-4 receptor/JAK1/STAT6 signaling pathway.

Progesterone Induced Blocking Factor Isoforms in Normal and Failed Murine Pregnancies

Progesterone induced blocking factor (PIBF) is required for successful pregnancy. Alternative splicing produces PIBF isoforms with different functions. The full-length (90 kDa) PIBF is involved in cell cycle regulation, whereas smaller secreted forms act as cytokines. We aim to examine the PIBF exon pattern and protein isoform profile in normal and failed murine pregnancies. Pregnant Balb/c mice were killed on gestation days 12-14 or 17-19. Normal and resorbed fetuses, placentae, and uterine tissue were used for RNA and protein analysis with RT-PCR and Western blot, respectively. Late pregnancy and resorption were associated with lower expression of the N-terminal exons, together with significantly reduced production of the full-length protein. Reduced production of the full-length PIBF protein might result in disturbed cell cycle regulation and dysregulated trophoblast invasion, while the absence of PIBF isoforms containing exon 2-4 coded sequences might lead to the loss of local immunosuppression.

Abstract 490: Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF).

Abstract Recently a sensitive ELISA assay been created to detect serum levels of the immunomodulatory protein, the PIBF. One of the 34 kDa forms of PIBF increases markedly after exposure to progesterone (P) and inhibits natural killer (NK) cytolytic activity by stabilizing perforin granules and causes of shift from thymic helper (TH)1 to TH2 dominance thus diminishing the activity of the cellular immune system. The 34 kDa form is secreted by gamma/delta T cells and is considered a vital step in preventing immune destruction of the fetal semi-allograft. Evaluation of mRNA for PIBF and PIBF protein found that 29 of 29 human leukemia cell lines over-expressed mRNA for PIBF but only 3 of the lines expressed the PIBF protein. Adding P to the culture up-regulated PIBF protein expression whereas adding the P receptor antagonist mifepristone caused down-regulation of PIBF expression. Mifepristone treatment has been demonstrated to show significant palliative effects on a wide variety of murine and human cancers many of which are not known to be P receptor positive. The hypothesized mechanism is that these tumors either secrete PIBF or direct secretion by gamma/delta T cells in the tumor microenvironment and thus inhibit NK cell cytolytic activity. The inhibition is abrogated by inhibiting P receptors and thus a local form of P secretion by the tumor is hypothesized. The objective of this study was to determine if mifepristone will only help inhibit cancer growth in those individuals who have an increased PIBF level present in their serum. An 81 year old woman with chronic lymphocytic leukemia (CLL) started more rapid advancement and became very symptomatic. She refused chemotherapy when she almost had a lethal complication to oral chlorambucil. She requested mifepristone. Her serum PIBF level of 34.9 ng/mL was in the same range as controls without cancer. However within a month of taking 200mg daily of mifepristone orally she showed a dramatic improvement in her CLL with her white blood cell count dropping from 28,000 to 8,000. Her platelet count increased from 40,000 to 240,000 and 2 lung nodules thought to represent possible primary lung cancer (but possibly nodules from her CLL) completely disappeared on repeat CT scan. Also her energy markedly improved and her persistent cough stopped. The serum PIBF did not decrease with mifepristone therapy - 48.3 ng/mL one month later. These data demonstrated that failure to see a marked increase in serum PIBF in a patient with cancer does not predict failure to respond to mifepristone. Though mifepristone has ameliorated murine leukemia in AKR mice this is the first case report of this drug improving human leukemia. The 34-36 kDa PIBF found in cytoplasm of cancer cells may either not be secreted in the serum or may be immunologically different from serum PIBF found after P exposure. Citation Format: Jerome H. Check, Ann DiAntonio, Diane Check, Alex Jaffe, Rachael Cohen, Mojirayo Sarumi, Maya Srivastava. Clinical improvement of symptomatic advancing chronic lymphocytic leukemia following mifepristone therapy despite normal serum levels of the immunomodulatory protein the progesterone induced blocking factor (PIBF). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2013-490

Endometrial expression of progesterone-induced blocking factor and galectins-1, -3, -9, and -3 binding protein in the luteal phase and early pregnancy in cattle

Progesterone-induced blocking factor (PIBF) and galectins modulate the maternal immune response during pregnancy. We hypothesized that the relative transcript abundance of the above genes would be different during the luteal phase/early pregnancy and would be affected by progesterone supplementation. To further test this, hypothesis protein expression analyses were carried out to evaluate the abundance and localization of LGALS9 and PIBF. Following estrus synchronization, heifers were inseminated (n = 140) or not (n = 70). Half the heifers in each status (cyclic or potentially pregnant) were randomly assigned to receive a progesterone-releasing intravaginal device (PRID) on day 3 after estrus, which elevated progesterone concentrations from day 3.5 to 8 (P < 0.05), resulting in four treatment groups: cyclic and pregnant heifers, each with normal and high progesterone. After confirmation of pregnancy status in inseminated animals, uterine tissue was collected on days 5, 7, 13, or 16 of the luteal phase of the cycle/pregnancy. Gene and protein expression was determined using Q-RT-PCR and IHC, respectively, on 5 heifers per treatment per time point (i.e., 80 in total). Progesterone concentrations did not affect expression of any of the genes (P > 0.05). LGALS9 and LGALS3BP were expressed at low levels in both cyclic and pregnant endometria until day 13. On day 16, expression increased only in the pregnant heifers (P < 0.0001). LGALS1 and LGALS3 decreased on day 7 (P < 0.0001) and remained low until day 16. Pregnancy had no effect on the expression of LGALS1, LGALS3, and PIBF. Additionally, LGALS9 and PIBF proteins were expressed in distinct uterine cell types. These results indicate that the galectins may be involved in uterine receptivity and/or implantation in heifers.

Abstract 785: The progesterone induced blocking factor, an immunomodulatory protein that may suppress natural killer cell activity in the tumor microenvironment, found to be a soluble protein possibly allowing the development of an enzyme-linked immunoabsorbent assay

Abstract A unique 34 kDa protein expressed by gamma delta thymic (T) cells during pregnancy suppresses natural killer (NK) cell activity and helps to shift the balance of thymic helper (TH) 1 to TH2 cytokine dominance. The mechanism involved in making this immunomodulaotry protein involves de novo induction of progesterone (P) receptors on gamma delta T cells by the allogeneic stimulus of the fetal semi-allograft and the interaction of the P receptors with a high concentration of P generated at the maternal fetal interface. There is evidence that certain cancer cells may use a similar mechanism to escape NK cell surveillance as evidenced by all 27 different human leukemia cells tested showing a very high level of messenger RNA for this immunomodulatory protein known as the progesterone induced blocking factor (PIBF). Furthermore adding the P receptor antagonist mifepristone down-regulated PIBF expression. PIBF may be synthesized by gamma delta T cells in the tumor microenvironment of solid tumors thus inhibiting NK cell immunosurveillance. Previous studies were performed using an immunocytochemistry technique using a polyclonal antibody because the PIBF protein was not purified. Recombinant DNA technology has allowed the production of a pure protein and thus the development of a monoclonal antibody. The present study was aimed at determining if PIBF is a soluble protein, which if it is could lead to the development of a much more practical and sensitive enzyme linked immunoabsorbent assay (ELISA). Serum samples were blindly assessed for the presence of PIBF using a novel sandwich ELISA we developed with full length (amino acid 1 to 757) recombinant human PIBF, rabbit polyclonal antibody to amino acid 1-300, and affinity purified goat polyclonal antibody to the internal region of PIBF conjugated to horseradish peroxidase. Color was developed using tetramethylbenzidine substrate stopped by 2N H2SO4 and read at 450 lambda. Bovine serum albumin served as negative control. Soluble PIBF was detected in several samples, with highest levels in pregnant patients at over 10 microgram/ml concentration. There were significant inter-individual differences. PIBF, does indeed also exist in a soluble form and can be detected in serum. With further refinement of our ELISA we aim to improve sensitivity and specificity, allowing for the simple measurement of PIBF on a rapid, high throughput basis, making it a more practical test to predict cancers that would benefit from progesterone receptor antagonist therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 785. doi:10.1158/1538-7445.AM2011-785

PIBF: The Double Edged Sword. Pregnancy and Tumor

The role of progesterone-dependent immunomodulation in the maintenance of normal pregnancy. In vitro and in vivo data on the effect that progesterone and its mediator progesterone-induced blocking factor (PIBF) exert on the immune functions of pregnant women are reviewed, together with clinical findings. Activated pregnancy lymphocytes express progesterone receptors, which enable progesterone to induce a protein called PIBF. PIBF increases Th2 type cytokine production by signaling via a novel type of IL-4 receptor and activating the Jak/STAT pathway. PIBF inhibits phosholipase A2, thus reduces prostaglandin synthesis. PIBF inhibits perforin release in human decidual lymphocytes and reduces the deleterious effect of high NK activity on murine pregnancy. PIBF production is a characteristic feature of normal human pregnancy, and its concentration is reduced in threatened pregnancies. PIBF mRNA and protein are expressed in a variety of malignant tumors. Inhibition of PIBF synthesis increases survival rates of leukemic mice. Progesterone-induced blocking factor is produced by pregnancy lymphocytes and also by malignant tumors. The PIBF-induced Th2-dominant immune response is favorable during pregnancy but might facilitate tumor growth by suppressing local antitumor immune responses.

Support for the hypothesis that successful immunotherapy of various cancers can be achieved by inhibiting a progesterone associated immunomodulatory protein

A hypothesis was proposed that cancer cells may utilize a pre-existing mechanism that pregnant mammals use to avoid natural killer cell immune surveillance of the fetus. The hypothesis suggested that those cancer cells that are able to proliferate may have found a way to cause the expression of the immunomodulatory protein known as the progesterone induced blocking factor (PIBF). The cancer cells could find an alternate pathway to make this protein that does not require progesterone secretion, or the cancer cells may actually utilize progesterone and thus make PIBF in a similar fashion to normal pregnancy. If the former mechanism was operational, then one could develop monoclonal antibody type therapy directed to this unique protein not needed for normal body functions. However, if the latter pathway involving progesterone secretion is operational, then there would be drugs already on the market, e.g., the progesterone receptor antagonist mifepristone that could be used to treat these cancers. In vitro data has shown that 100% of human leukemia cell lines express mRNA for the PIBF protein. Some leukemia cell lines have been found that actually express the PIBF protein. In fact adding progesterone to the culture media upregulated PIBF protein expression and mifepristone inhibited it. Controlled studies in various murine spontaneous cancers not known to be associated with progesterone receptors showed increased length and quality of life following mifepristone therapy. Anecdotal improvement in advanced widely metastatic human cancers has also been found. Thus there is now experimental data to support this hypothesis and a new door to a completely different type of cancer therapy has been opened.

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48 kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1–18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90 kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34 kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.

Expression and modulation of progesterone induced blocking factor (PIBF) and innate immune factors in human leukemia cell lines by progesterone and mifepristone

Progesterone (P), required for successful pregnancy, influences autoimmune, infectious, and malignant diseases via adaptive and innate immune effects. P induces NK inhibitor progesterone induced blocking factor (PIBF) in CD8+ T cells. PIBF isoforms could permit solid tumor immune escape. Expression and modulation of PIBF and innate immune proteins by P in leukemia cells and leukocyte subpopulations have not been reported. Ten T, seven myeloid, six B, five epithelial, fibroblast BG9, G-CSF mobilized CD34+ stem cells, and peripheral blood mononuclear cells were screened for PIBF mRNA by RT-PCR, and protein by immunohistochemistry in SRIK-NKL, MOT, U937, HL60, R-CLL, MD-E, 729pH6neo, SRIH-B(ATL), SRIK-B(T-PLL), and MeWo. Cell lines expressing PIBF and exemplifying myeloid/monoblast, natural killer/T, and B lineages were cultured with and without 0.5 – 5 µM P or 0.5 – 0.05 µM mifepristone (RU486) for 24 h. Subsequently they were examined for changes in the expression of mRNA by RT-PCR and protein by immunohistochemistry for PIBF and some innate immune factors. All cells expressed PIBF mRNA; protein only in four (SRIK-NKL, U937, SRIK-B(T-PLL) and HL60) out of 10 cell lines tested. P increased and RU486 decreased PIBF in U937, SRIK-B(T-PLL) and SRIK-NKL. P upregulated TLR-4 in U937, and HNP1 – 3, LL-37, IRAK-2, and IRAK-4 in multiple lines and RU486 down regulated these. PIBF may be used by some leukemias to evade immune surveillance and is a potential therapeutic target. P may impact infection and autoimmunity via effects on LPS receptor, TLR signaling, and antimicrobial peptides.

PIBF (progesterone induced blocking factor) is overexpressed in highly proliferating cells and associated with the centrosome.

PIBF was previously identified as a 34 kDa immunomodulatory molecule secreted by pregnancy lymphocytes and is thought to play a crucial role in preventing rejection of the embryo by the maternal immune response. Recent data suggested that PIBF protein was also expressed by the progesterone receptor (PR) positive MCF-7 breast tumor cell line. Therefore our study was designed to analyze the expression of PIBF in malignant cell lines and primary tumors both at the mRNA and protein levels. RNA expression analyses of several human cell lines with different tissue origin and paired human tumor/normal tissues, as well as of several PR+ and PR- breast tumors revealed that PIBF mRNA was overexpressed in highly proliferating cells independent of the presence of PR. In addition to the full-length PIBF mRNA encoding for a 90 kDa protein, several alternatively spliced species were detected, all resulting from perfect exon skipping. The most frequently identified splice variant is predicted to encode for an approximately 35 kDa protein. Immunofluorescence microscopy revealed a centrosomal localization for the full-length PIBF, while the 35 kDa form showed a diffuse cytoplasmic staining. These data, together with the identification of the PIBF gene in the chromosomal region associated with breast cancer susceptibility, reveal a strong parallel with known tumor suppressor proteins, such as BRCA1 and p53 having the same centrosomal localization. Given the notion that a number of proteins shown to be involved in tumorigenesis are associated with the centrosome and disturbed centrosome function causes unequal segregation of chromosomes, studies to evaluate whether or not PIBF that is highly expressed in tumors is directly involved in tumorigenesis are thus warranted.

Modulation and function of progesterone induced blocking factor (PIBF) in U937 and T-PLL cell lines

Rationale Placental CD8+ T cells exposed to progesterone produce progesterone induced blocking factor (PIBF), a 34-kDa protein that decreases natural killer cytotoxicity and prevents fetal rejection. Tumor cells could utilize PIBF for immune escape. Methods Cell lines were screened for PIBF production. U937 (monoblastic leukemia) and T-PLL (non-leukemic B-lymphocyte precursor) demonstrated high expression and were chosen for further study. Cells were cultured in RPMI 1640, 5% fetal bovine serum, 1% L-glutamine, penicillin-streptomycin until above 95% viability and count 0.5 × 106 cells/ml, and plated without and with physiologic progesterone concentrations of 5 μM and 0.5 μM, or antagonist mifepristone at 0.5 μM and 0.05 μM for 12, 24, and 48 hours. RNA was extracted (Qiagen) and semiquantitative RT-PCR conducted using primers designed with website http://www.ncbi.nih.gov and Primer 3 program for PIBF, Toll-like receptors 2, 4, 5, 7, NOD-2, IKB-α, MyD88, IRAK -1, Tollip, and LL-37. Based on RT-PCR, immunohistochemistry was performed with anti-TLR-4, TLR-2, LL-37, and PIBF antibodies and DAKO EnVision Plus staining kits. Results PIBF mRNA and protein were upregulated with progesterone and downregulated by mifepristone in both cell lines. Mifepristone decreased LL-37 protein in T-PLL. TLR4 mRNA and protein, and HNP 1-3 mRNA increased with higher amounts of progesterone in U937. No changes were observed in the other genes tested. Conclusions PIBF is expressed and modulated by progesterone in B and monocyte lineages in addition to T cells. Further investigation must differentiate whether effects on antimicrobial peptides and toll-like receptors are via progesterone receptor or through altered autocrine actions of PIBF.