Sustained delivery of celecoxib from nanoparticles embedded in hydrogel injected into the biopsy cavity to prevent biopsy-induced breast cancer metastasis.

PURPOSE: We have previously reported that protracted Cyclooxygenase-2 (COX-2) activity in bone marrow-derived cells (BMDCs) infiltrating into biopsy wounds adjacent to the biopsy cavity of breast tumors in mice promotes M2-shift of macrophages and pro-metastatic changes in cancer cells, effects which were suppressed by oral administration of COX-2 inhibitors. Thus, local control of COX-2 activity in the biopsy wound may mitigate biopsy-induced pro-metastatic changes. METHODS: A combinatorial delivery system-thermosensitive biodegradable poly(lactic acid) hydrogel (PLA-gel) incorporating celecoxib-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Cx-NP/PLA-gel)-was injected into the biopsy cavity of Py230 murine breast tumors to achieve local control of COX-2 activity in the wound stroma. RESULTS: A single intra-biopsy cavity injection of PLA-gel loaded with rhodamine-encapsulated nanoparticles (NPs) showed sustained local delivery of rhodamine preferentially to infiltrating BMDCs with minimal to no rhodamine uptake by the reticuloendothelial organs in mice. Moreover, significant reductions in M2-like macrophage density, cancer cell epithelial-to-mesenchymal transition, and blood vessel density were observed in response to a single intra-biopsy cavity injection of Cx-NP/PLA-gel compared to PLA-gel loaded with NPs containing no payload. Accordingly, intra-biopsy cavity injection of Cx-NP/PLA-gel led to significantly fewer metastatic cells in the lungs than control-treated mice. CONCLUSION: This study provides evidence for the feasibility of sustained, local delivery of payload preferential to BMDCs in the wound stroma adjacent to the biopsy cavity using a combinatorial delivery system to reduce localized inflammation and effectively mitigate breast cancer cell dissemination.

Needle biopsy accelerates pro-metastatic changes and systemic dissemination in breast cancer: Implications for mortality by surgery delay.

Increased breast cancer (BC) mortality risk posed by delayed surgical resection of tumor after diagnosis is a growing concern, yet the underlying mechanisms remain unknown. Our cohort analyses of early-stage BC patients reveal the emergence of a significantly rising mortality risk when the biopsy-to-surgery interval was extended beyond 53 days. Additionally, histology of post-biopsy tumors shows prolonged retention of a metastasis-permissive wound stroma dominated by M2-like macrophages capable of promoting cancer cell epithelial-to-mesenchymal transition and angiogenesis. We show that needle biopsy promotes systemic dissemination of cancer cells through a mechanism of sustained activation of the COX-2/PGE2/EP2 feedforward loop, which favors M2 polarization and its associated pro-metastatic changes but are abrogated by oral treatment with COX-2 or EP2 inhibitors in estrogen-receptor-positive (ER+) syngeneic mouse tumor models. Therefore, we conclude that needle biopsy of ER+ BC provokes progressive pro-metastatic changes, which may explain the mortality risk posed by surgery delay after diagnosis.

High PD-L2 Predicts Early Recurrence of ER-Positive Breast Cancer.

PURPOSE: T-cell-mediated cytotoxicity is suppressed when programmed cell death-1 (PD-1) is bound by PD-1 ligand-1 (PD-L1) or PD-L2. Although PD-1 inhibitors have been approved for triple-negative breast cancer, the lower response rates of 25%-30% in estrogen receptor-positive (ER+) breast cancer will require markers to identify likely responders. The focus of this study was to evaluate whether PD-L2, which has higher affinity than PD-L1 for PD-1, is a predictor of early recurrence in ER+ breast cancer. METHODS: PD-L2 protein levels in cancer cells and stromal cells of therapy-naive, localized or locoregional ER+ breast cancers were measured retrospectively by quantitative immunofluorescence histocytometry and correlated with progression-free survival (PFS) in the main study cohort (n = 684) and in an independent validation cohort (n = 273). All patients subsequently received standard-of-care adjuvant therapy without immune checkpoint inhibitors. RESULTS: Univariate analysis of the main cohort revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (hazard ratio [HR], 1.8; 95% CI, 1.3 to 2.6; P = .001), which was validated in an independent cohort (HR, 2.3; 95% CI, 1.1 to 4.8; P = .026) and remained independently predictive after multivariable adjustment for common clinicopathological variables (HR, 2.0; 95% CI, 1.4 to 2.9; P < .001). Subanalysis of the ER+ breast cancer patients treated with adjuvant chemotherapy (n = 197) revealed that high PD-L2 levels in cancer cells associated with short PFS in univariate (HR, 2.5; 95% CI, 1.4 to 4.4; P = .003) and multivariable analyses (HR, 3.4; 95% CI, 1.9 to 6.2; P < .001). CONCLUSION: Up to one third of treatment-naive ER+ breast tumors expressed high PD-L2 levels, which independently predicted poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Collectively, these data warrant studies to gain a deeper understanding of PD-L2 in the progression of ER+ breast cancer and may provide rationale for immune checkpoint blockade for this patient group.

Co-targeting KRAS G12C and EGFR reduces both mutant and wild-type RAS-GTP.

The combination of KRAS G12C inhibitors with EGFR inhibitors has reproducibly been shown to be beneficial. Here, we identify another benefit of this combination: it effectively inhibits both wild-type and mutant RAS. We believe that targeting both mutant and wild-type RAS helps explain why this combination of inhibitors is effective.

NSG-Pro mouse model for uncovering resistance mechanisms and unique vulnerabilities in human luminal breast cancers.

Most breast cancer deaths are caused by estrogen receptor-α­positive (ER+) disease. Preclinical progress is hampered by a shortage of therapy-naïve ER+ tumor models that recapitulate metastatic progression and clinically relevant therapy resistance. Human prolactin (hPRL) is a risk factor for primary and metastatic ER+ breast cancer. Because mouse prolactin fails to activate hPRL receptors, we developed a prolactin-humanized Nod-SCID-IL2Rγ (NSG) mouse (NSG-Pro) with physiological hPRL levels. Here, we show that NSG-Pro mice facilitate establishment of therapy-naïve, estrogen-dependent PDX tumors that progress to lethal metastatic disease. Preclinical trials provide first-in-mouse efficacy of pharmacological hPRL suppression on residual ER+ human breast cancer metastases and document divergent biology and drug responsiveness of tumors grown in NSG-Pro versus NSG mice. Oncogenomic analyses of PDX lines in NSG-Pro mice revealed clinically relevant therapy-resistance mechanisms and unexpected, potently actionable vulnerabilities such as DNA-repair aberrations. The NSG-Pro mouse unlocks previously inaccessible precision medicine approaches for ER+ breast cancers.

Sensitive and Specific Immunohistochemistry Protocol for Nucleocapsid Protein from All Common SARS-CoV-2 Virus Strains in Formalin-Fixed, Paraffin Embedded Tissues.

Human coronavirus disease 2019 (COVID-19) is a life-threatening and highly contagious disease caused by coronavirus SARS-CoV-2. Sensitive and specific detection of SARS-CoV-2 viral proteins in tissues and cells of COVID-19 patients will support investigations of the biologic behavior and tissue and cell tropism of this virus. We identified commercially available affinity-purified polyclonal antibodies raised against nucleocapsid and spike proteins of SARS-CoV-2 that provide sensitive and specific detection of the virus by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Two immunohistochemistry protocols are presented that are mutually validated by the matched detection patterns of the two distinct viral antigens in virus-infected cells within autopsy lung tissue of COVID-19 deceased patients. Levels of nucleocapsid protein in the lungs of COVID-19 decedents, as measured by quantitative histo-cytometry of immunohistochemistry images, showed an excellent log-linear relationship with levels of viral nucleocapsid RNA levels, as measured by qRT-PCR. Importantly, since the nucleocapsid protein sequence is conserved across all known viral strains, the nucleocapsid immunohistochemistry protocol is expected to recognize all common variants of SARS-CoV-2. Negative controls include autopsy lung tissues from patients who died from non-COVID-19 respiratory disease and control rabbit immunoglobulin. Sensitive detection of SARS-CoV-2 in human tissues will provide insights into viral tissue and cell distribution and load in patients with active infection, as well as provide insight into the clearance rate of virus in later COVID-19 disease stages. The protocols are also expected to be readily transferable to detect SARS-CoV-2 proteins in tissues of experimental animal models or animals suspected to serve as viral reservoirs.

Diffuse interstitial pneumonia-like/macrophage activation syndrome-like changes in patients with COVID-19 correlate with length of illness.

OBJECTIVES: Assess the pathologic changes in the lungs of COVID-19 decedents and correlate these changes with demographic data, clinical course, therapies, and duration of illness. METHODS: Lungs of 12 consecutive COVID-19 decedents consented for autopsy were evaluated for gross and histopathologic abnormalities. A complete Ghon "en block" dissection was performed on all cases; lung weights and gross characteristics recorded. Immunohistochemical studies were performed to characterize lymphocytic infiltrates and to assess SARS-CoV-2 capsid protein. RESULTS: Two distinct patterns of pulmonary involvement were identified. Three of 12 cases demonstrated a predominance of acute alveolar damage (DAD) while 9 of 12 cases demonstrated a marked increase in intra-alveolar macrophages in a fashion resembling desquamative interstitial pneumonia or macrophage activation syndrome (DIP/MAS). Two patterns were correlated solely with a statistically significant difference in the duration of illness. The group exhibiting DAD had duration of illness of 5.7 days while the group with DIP/MAS had duration of illness of 21.5 days (t-test p = 0.014). CONCLUSIONS: The pulmonary pathology of COVID-19 patients demonstrates a biphasic pattern, an acute phase demonstrating DAD changes while the patients with a more prolonged course exhibit a different pattern that resembles DIP/MAS-like pattern. The potential mechanisms and clinical significance are discussed.

Loss of Nuclear Localized Parathyroid Hormone-Related Protein in Primary Breast Cancer Predicts Poor Clinical Outcome and Correlates with Suppressed Stat5 Signaling.

PURPOSE: Parathyroid hormone-related protein (PTHrP) is required for normal mammary gland development and biology. A PTHLH gene polymorphism is associated with breast cancer risk, and PTHrP promotes growth of osteolytic breast cancer bone metastases. Accordingly, current dogma holds that PTHrP is upregulated in malignant primary breast tumors, but solid evidence for this assumption is missing. EXPERIMENTAL DESIGN: We used quantitative IHC to measure PTHrP in normal and malignant breast epithelia, and correlated PTHrP levels in primary breast cancer with clinical outcome. RESULTS: PTHrP levels were markedly downregulated in malignant compared with normal breast epithelia. Moreover, low levels of nuclear localized PTHrP in cancer cells correlated with unfavorable clinical outcome in a test and a validation cohort of breast cancer treated at different institutions totaling nearly 800 cases. PTHrP mRNA levels in tumors of a third cohort of 737 patients corroborated this association, also after multivariable adjustment for standard clinicopathologic parameters. Breast cancer PTHrP levels correlated strongly with transcription factors Stat5a/b, which are established markers of favorable prognosis and key mediators of prolactin signaling. Prolactin stimulated PTHrP transcript and protein in breast cancer cell lines in vitro and in vivo, effects mediated by Stat5 through the P2 gene promoter, producing transcript AT6 encoding the PTHrP 1-173 isoform. Low levels of AT6, but not two alternative transcripts, correlated with poor clinical outcome. CONCLUSIONS: This study overturns the prevailing view that PTHrP is upregulated in primary breast cancers and identifies a direct prolactin-Stat5-PTHrP axis that is progressively lost in more aggressive tumors.

GHR/PRLR Heteromultimer Is Composed of GHR Homodimers and PRLR Homodimers.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane cytokine receptors. Each prehomodimerizes and ligand binding activates Janus Kinase 2 (JAK2)-signal transducer and activator of transcription (STAT) signaling pathways by inducing conformational changes within receptor homodimers. In humans, GHR is activated by GH, whereas PRLR is activated by both GH and PRL. We previously devised a split luciferase complementation assay, in which 1 receptor is fused to an N-terminal luciferase (Nluc) fragment, and the other receptor is fused to a C-terminal luciferase (Cluc) fragment. When receptors approximate, luciferase activity (complementation) results. Using this assay, we reported ligand-independent GHR-GHR complementation and GH-induced complementation changes characterized by acute augmentation above basal signal, consistent with induction of conformational changes that bring GHR cytoplasmic tails closer. We also demonstrated association between GHR and PRLR in T47D human breast cancer cells by coimmunoprecipitation, suggesting that, in addition to forming homodimers, these receptors form hetero-assemblages with functional consequences. We now extend these analyses to examine basal and ligand-induced complementation of coexpressed PRLR-Nluc and PRLR-Cluc chimeras and coexpressed GHR-Nluc and PRLR-Cluc chimeras. We find that PRLR-PRLR and GHR-PRLR form specifically interacting ligand-independent assemblages and that either GH or PRL augments PRLR-PRLR complementation, much like the GH-induced changes in GHR-GHR dimers. However, in contrast to the complementation patterns for GHR-GHR or PRLR-PRLR homomers, both GH and PRL caused decline in luciferase activity for GHR-PRLR heteromers. These and other data suggest that GHR and PRLR associate in complexes comprised of GHR-GHR/PRLR-PRLR heteromers consisting of GHR homodimers and PRLR homodimers, rather than GHR-PRLR heterodimers.

Aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling in endometriosis.

STUDY QUESTION: Are STAT3 signaling molecules differentially expressed in endometriosis? SUMMARY ANSWER: Levels of phospho-STAT3 and HIF1A, its downstream signaling molecule, are significantly higher in eutopic endometrium from women with endometriosis when compared with women without the disease. WHAT IS KNOWN ALREADY: Endometriosis is an estrogen-dependent inflammatory condition. Interleukin 6 (IL-6) is an inflammatory survival cytokine known to induce prolonged activation of STAT3 via association with the IL-6 receptor. STUDY DESIGN, SIZE, DURATION: Cross-sectional measurements of STAT3 and HIF1A protein levels in eutopic endometrium from women with endometriosis versus those without. PARTICIPANTS/MATERIALS, SETTING, METHODS: Levels of phospho-STAT3 (pSTAT3) and HIF1A were examined in the endometrium of patients with and without endometriosis as well as in a non-human primate animal model using western blot and immunohistochemical analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Levels of pSTAT3 were significantly higher in the eutopic endometrium from women with endometriosis when compared with women without the disease in both the proliferative and secretory phases. HIF1A is known to be stabilized by STAT3 and IL-6. Our immunohistochemistry results show abundant HIF1A expression within the eutopic endometrial epithelial cells of women with endometriosis. Furthermore, pSTAT3 and HIF1A proteins are co-localized in endometriosis. This aberrant activation of pSTAT3 and HIF1A is confirmed by sequential analysis of eutopic endometrium using a baboon animal model of induced endometriosis. Lastly, we confirmed this IL-6 induction of both STAT3 phosphorylation and HIF1A mRNA expression in Ishikawa human endometrial adenocarcinoma cell line. LIMITATIONS, REASONS FOR CAUTION: Ishikawa cancer cell line was used to study a benign disease. The peritoneal fluid contains various inflammatory cytokines in addition to IL-6 and so it is possible that other cytokines may affect the activity and expression of STAT3 signaling molecules. WIDER IMPLICATIONS OF THE FINDINGS: Our results imply that aberrant activation of STAT3 signaling plays an important role in the pathogenesis of endometriosis. Our findings could progress in our understanding of the etiology and pathophysiology of endometriosis and potential therapeutic interventions by targeted pharmacological. STUDY FUNDING/COMPETING INTERESTS: This work was supported by NIH R01 HD067721 (to S.L.Y and B.A.L) and NIH R01 HD057873 and American Cancer Society Research Grant RSG-12-084-01-TBG (to J.-W.J.). There are no conflicts of interest.

Dynamic analysis of GH receptor conformational changes by split luciferase complementation.

The transmembrane GH receptor (GHR) exists at least in part as a preformed homodimer on the cell surface. Structural and biochemical studies suggest that GH binds GHR in a 1:2 stoichiometry to effect acute GHR conformational changes that trigger the activation of the receptor-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signaling. Despite information about GHR-GHR association derived from elegant fluorescence resonance energy transfer/bioluminescence resonance energy transfer studies, an assessment of the dynamics of GH-induced GHR conformational changes has been lacking. To this end, we used a split luciferase complementation assay that allowed detection in living cells of specific ligand-independent GHR-GHR interaction. Furthermore, GH treatment acutely augmented complementation of enzyme activity between GHRs fused, respectively, to N- and C-terminal fragments of firefly luciferase. Analysis of the temporal pattern of GH-induced complementation changes, pharmacological manipulation, genetic alteration of JAK2 levels, and truncation of the GHR intracellular domain (ICD) tail suggested that GH acutely enhances proximity of the GHR homodimer partners independent of the presence of JAK2, phosphorylation of GHR-luciferase chimeras, or an intact ICD. However, subsequent reduction of complementation requires JAK2 kinase activity and the ICD tail. This conclusion is in contrast to existing models of the GHR activation process.

The role of prolactin receptor in GH signaling in breast cancer cells.

GH and prolactin (PRL) are structurally related hormones that exert important effects in disparate target tissues. Their receptors (GHR and PRLR) reside in the cytokine receptor superfamily and share signaling pathways. In humans, GH binds both GHR and PRLR, whereas PRL binds only PRLR. Both hormones and their receptors may be relevant in certain human and rodent cancers, including breast cancer. GH and PRL promote signaling in human T47D breast cancer cells that express both GHR and PRLR. Furthermore, GHR and PRLR associate in a fashion augmented acutely by GH, even though GH primarily activates PRLR, rather than GHR, in these cells. To better understand PRLR's impact, we examined the effects of PRLR knockdown on GHR availability and GH sensitivity in T47D cells. T47D-ShPRLR cells, in which PRLR expression was reduced by stable short hairpin RNA (shRNA) expression, were compared with T47D-SCR control cells. PRLR knockdown decreased the rate of GHR proteolytic turnover, yielding GHR protein increase and ensuing sensitization of these cells to GHR signaling events including phosphorylation of GHR, Janus kinase 2, and signal transducer and activator of transcription 5 (STAT5). Unlike in T47D-SCR cells, acute GH signaling in T47D-ShPRLR cells was not blocked by the PRLR antagonist G129R but was inhibited by the GHR-specific antagonist, anti-GHR(ext-mAb). Thus, GH's use of GHR rather than PRLR was manifested when PRLR was reduced. In contrast to acute effects, GH incubation for 2 h or longer yielded diminished STAT5 phosphorylation in T47D-ShPRLR cells compared with T47D-SCR, a finding perhaps explained by markedly greater GH-induced GHR down-regulation in cells with diminished PRLR. However, when stimulated with repeated 1-h pulses of GH separated by 3-h washout periods to more faithfully mimic physiological GH pulsatility, T47D-ShPRLR cells exhibited greater transactivation of a STAT5-responsive luciferase reporter than did T47D-SCR cells. Our data suggest that PRLR's presence meaningfully affects GHR use in breast cancer cells.

Stromal-epithelial interactions modulate cross-talk between prolactin receptor and HER2/Neu in breast cancer.

Prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been developed, which indirectly inhibits the tyrosine phosphorylation of HER2 (p-HER2) in human breast cancer cell lines. In this study, we investigate the effects of cancer-associated fibroblasts (CAFs) upon this molecular cross-talk using tumor cells and CAFs derived from spontaneous mammary tumors of female MMTV-neu transgenic mice. Tumors were resected and cultured as small tumor chunks (~3 mm3) or were cultured in monolayer. G129R reduced tyrosine phosphorylation of Neu (p-Neu) in a dose-dependent manner (IC50~10 µg/ml) in tumor chunks, but had no effect on primary tumor epithelial cells grown in monolayer. Direct co-culture of mouse or human tumor epithelial cell lines with CAFs restored the epithelial cells' response to G129R, similar to that observed in mouse tumor chunks. The addition of PRL, as expected, induced p-Neu in both the tumor chunk and co-culture models. The inhibitory effect of G129R was absent when CAFs were physically separated from mouse tumor epithelial cells using a transwell system, or when CAFs were replaced with normal fibroblasts in direct co-culture with human or mouse tumor epithelial cells. In vivo, G129R reduced p-Neu levels in primary mammary tumors of mice in a time- and dose-dependent manner. In conclusion, CAFs play a critical role in bridging the cross-talk between PRL and HER2/Neu in both mouse and human models of breast cancer. The inhibitory effects of G129R on p-Neu and on tumor growth are dependent upon interactions of tumor epithelial cells with CAFs.

Growth hormone signaling in human T47D breast cancer cells: potential role for a growth hormone receptor-prolactin receptor complex.

GH receptor (GHR) and prolactin (PRL) receptor (PRLR) are structurally similar cytokine receptor superfamily members that are highly conserved among species. GH has growth-promoting and metabolic effects in various tissues in vertebrates, including humans. PRL is essential for regulation of lactation in mammals. Recent studies indicate that breast tissue bears GHR and PRLR and that both GH and PRL may impact development or behavior of breast cancer cells. An important facet of human GH (hGH) and human PRL (hPRL) biology is that although hPRL interacts only with hPRLR, hGH binds well to both hGHR and hPRLR. Presently, we investigated potential signaling effects of both hormones in the estrogen receptor- and progesterone receptor-positive human T47D breast cancer cell line. We found that this cell type expresses ample GHR and PRLR and responds well to both hGH and hPRL, as evidenced by activation of the Janus kinase 2/signal transducer and activator of transcription 5 pathway. Immunoprecipitation studies revealed specific GHR-PRLR association in these cells that was acutely enhanced by GH treatment. Although GH caused formation of disulfide-linked and chemically cross-linked GHR dimers in T47D cells, GH preferentially induced tyrosine phosphorylation of PRLR rather than GHR. Notably, both a GHR-specific ligand antagonist (B2036) and a GHR-specific antagonist monoclonal antibody (anti-GHR(ext-mAb)) failed to inhibit GH-induced signal transducer and activator of transcription 5 activation. In contrast, although the non-GHR-specific GH antagonist (G120R) and the PRL antagonist (G129R) individually only partially inhibited GH-induced activation, combined treatment with these two antagonists conferred greater inhibition than either alone. These data indicate that endogenous GHR and PRLR associate (possibly as a GHR-PRLR heterodimer) in human breast cancer cells and that GH signaling in these cells is largely mediated by the PRLR in the context of both PRLR-PRLR homodimers and GHR-PRLR heterodimers, broadening our understanding of how these related hormones and their related receptors may function in physiology and pathophysiology.

Improving the pharmacokinetics/pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding peptide.

To prolong the circulation half-life of human prolactin (hPRL), human GH (hGH), and their competitive antagonists, hPRL-G129R and hGH-G120R, we examined the effects of fusing a serum albumin-binding peptide (SA20) to their amino- or carboxyl-terminus. Fusion of the SA20 peptide to the amino-terminus of the ligands was less detrimental upon their ability to induce or inhibit signal transduction and cell proliferation in vitro than fusion to the carboxyl-terminus. Pharmacokinetic (PK) studies in mice revealed that the half-life of SA20-hPRL and SA20-hGH was prolonged and their clearance was reduced in comparison with hPRL and hGH. Pharmacodynamic (PD) studies in 8-week-old female mice revealed that lobuloalveolar development in mammary glands was greater in all three groups (daily, every 2 days, or every third day over a 12-day period) of mice treated with SA20-hPRL (4 mg/kg) compared with hPRL (3.59 mg/kg). Similarly, daily administration (i.p.) of SA20-hGH (8 mg/kg) or hGH (7.15 mg/kg) to 23-day-old female mice over a 40-day period revealed the superiority of SA20-hGH over hGH as measured by weight gain, body length, and lobuloalveolar development in the mammary glands. These findings indicate that SA20 modification of hPRL, hGH, and their respective antagonists improves their PK/PD properties.

Combination therapy using three novel prolactin receptor antagonist-based fusion proteins effectively inhibits tumor recurrence and metastasis in HER2/neu transgenic mice.

Previously, prolactin receptor antagonist (G129R)- based fusion proteins were developed including G129R fusions with an angiogenesis inhibitor (endostatin), an immune system modulator (interleukin 2), and a modified truncated cytotoxin (PE38KDEL). Each fusion protein was designed to target the PRLR-positive cells via the G129R moiety and at the same time attack a hallmark common to cancer cells via the second moiety. In this study, we tested the efficacy of the three fusion proteins as a combination therapy in an aggressive but clinically relevant mouse tumor model. To test the feasibility and to optimize a treatment regimen, allografts of a mammary carcinoma cell line (McNeuA) derived from an MMTV-neu transgenic mouse were first used. Growth of the allografts was significantly retarded by regimens which combined all three fusion proteins. In addition, a significant increase in cytotoxic CD8+ T cells was observed within the tumors of the combination treated groups. After establishing the dosing regimen, two doses of cocktail treatment (low and high doses administered twice weekly) along with individual component controls were administered to female MMTV-neu transgenic mice after surgical removal of a naturally occurring tumor. The average tumor recurrence time was significantly delayed in both low and high combination treatment groups in comparison to the no treatment control group (34, 50 and 18 days, respectively). The total number of lung metastases was also significantly decreased in both combination treatment groups. In conclusion, using G129R-based fusion proteins to target mammary carcinomas and to tackle multiple hallmarks of cancer at the same time was an effective strategy for treating HER2-postive mammary cancer in this mouse tumor model.

Activation of growth hormone receptors by growth hormone and growth hormone antagonist dimers: insights into receptor triggering.

GH binds dimerized GH receptors (GHRs) to form a trimolecular complex and induces downstream signaling events. The mechanism by which GH binding converts the inactive predimerized GHR to its active signaling conformation is uncertain. GH has no axis of symmetry. Its interaction with GHR is mediated by two asymmetric binding sites on GH, each with distinct affinity. Site 1 is of high affinity and is thought to mediate the first binding step. Mutation of binding site 2 (as in the human GH mutant, G120R) disrupts the second binding but leaves site 1 binding intact. G120R is a GH antagonist; it binds only one GHR and thus fails to signal, and it prevents productive GHR binding by normal GH. We previously demonstrated that prolactin receptor signaling was achieved by a dimeric version of a prolactin antagonist. We now employ assays of cellular signaling and receptor conformational changes to examine whether GH molecules harboring two site 1 regions can trigger GHR activation. We used recombinantly produced GH-GH and G120R-G120R dimers in which monomers in tandem are connected by a short linker peptide. Rabbit GHR-expressing human fibrosarcoma cells (C14) were treated with GH, G120R, GH-GH, or G120R-G120R. As expected, GH and GH-GH, but not G120R, induced GHR disulfide linkage, as assessed by anti-GHR blotting of cell extracts resolved by SDS-PAGE under nonreducing conditions. Disulfide linkage of GHRs reflects attainment of the active signaling conformation. Likewise, GH and GH-GH, but not G120R, caused Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) activation. Notably, G120R-G120R, despite its lack of an intact site 2 in either dimer partner, also promoted GHR disulfide linkage and JAK2 and STAT5 activation, albeit less potently than either GH or GH-GH. Time-course responses of the three agonists were similar in terms of JAK2 and STAT5 activation. Pretreatment of cells with our conformation-sensitive inhibitory monoclonal antibody, anti-GHR ext-mAb, prevented ligand-induced receptor activation for all three agonists. GHR was also rendered less immunoprecipitable by anti-GHR ext-mAb after treatment with these agonists. These results are important in that they indicate that a ligand with two intact binding sites 1 causes GHR to adopt similar conformational changes as does GH and thus triggers activation of JAK2 and downstream signaling. Furthermore, we infer that there is substantial flexibility in the GHR extracellular domain, such that it productively accommodates GH dimers that are much larger than GH.

Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells.

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

Two wrongs can make a right: dimers of prolactin and growth hormone receptor antagonists behave as agonists.

Prolactin (PRL) and GH have two distinct binding sites (site 1 with high affinity; site 2 with low affinity) that each interact with a PRL receptor (PRLR) to form a functional receptor dimer that activates signal transduction. The G129R mutation in PRL and the G120R mutation in GH disrupt the structural integrity of site 2 such that the ligands retain the ability to bind to the first receptor with high affinity, but act as receptor antagonists. In this study, we examined the ability of monomeric and dimeric forms of these ligands, human (h) PRL and hGH, and their antagonists (hPRL-G129R and hGH-G120R) to 1) bind to PRLRs; 2) induce conformational changes in PRLRs; 3) activate signaling pathways associated with the PRLR; and 4) mediate cell proliferation in vitro. In contrast to monomeric hPRL-G129R, homodimeric hPRL-G129R induced PRLR dimerization; activated Janus family of tyrosine kinases 2/signal transducer and activator of transcription 5, Ras/Raf/MAPK kinase/Erk, and phosphatidylinositol 3-kinase/Akt signaling; and stimulated Nb2 cell proliferation. Similarly, homodimeric hGH-G120R was able to mediate signaling via the PRLR and to stimulate Nb2 cell proliferation. These experiments demonstrate that a ligand must have two functional binding sites, but that these may be site 1 plus site 2 or two site 1's, to elicit receptor-mediated signal transduction. The size of the ligand plays less of a role in receptor activation, suggesting that the extracellular portion of the PRLR (and possibly the GH receptor) is rather flexible and can accommodate larger ligands. These findings may have implications for designing multifunctional therapeutics that target this class of cytokine receptors.

The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.