Hypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

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Teriparatide, recombinant parathyroid hormone (PTH[1‐34]), and abaloparatide, an analogue of PTH related‐peptide (PTHrP[1‐34]), are both anabolic medications for osteoporosis that target the PTH receptor PTH1R. PTH1R is a G protein–coupled receptor, and the stimulatory Gs protein is an important mediator of the anabolic actions of PTH1R activation in bone. We have published that mice lacking the α subunit of Gs in osteoprogenitors do not increase bone mass in response to PTH(1‐34). Unexpectedly, however, PTH(1‐34) still increases osteoblast numbers and bone formation rate in male mice, suggesting that PTH1R may have both Gs‐dependent and ‐independent actions in bone. Here we examine the role of Gs signaling in the anabolic actions of abaloparatide. We find that abaloparatide increases bone formation in male mice with postnatal deletion of G s α in Osx‐expressing osteoprogenitors (P‐G s α OsxKO mice) but not in female P‐G s α OsxKO mice. Therefore, abaloparatide has anabolic effects on bone in male but not female mice that appear to be independent of Gs‐mediated signaling. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

As tumors outgrow their blood supply, they frequently become hypoxic, activating hypoxia-inducible factor (HIF) signaling. Previous studies suggest that HIF signaling in breast cancer cells promotes lung dissemination in genetic models and bone colonization following intracardiac inoculation, but the impact of HIF signaling in the primary tumor on spontaneous dissemination to bone has never been evaluated. Thus, we hypothesized that Hif1α or Hif2α deletion in the primary tumor would reduce spontaneous dissemination to lung and bone, while deletion of Vhl (resulting in constitutive HIF signaling) would increase dissemination to distant metastatic sites. To test this, we generated MMTV-Cre.Hif1αf/f.PyMT, MMTV-Cre.Hif2αf/f.PyMT, and MMTV-Cre.Vhlf/f.PyMT mice, which spontaneously develop mammary carcinomas with conditional deletion of Hif1a, Hif2a, or Vhl, respectively. Littermate and cousin controls were used for all experiments. All mice were euthanized when the largest tumor reached 1cm in any dimension. Tumor growth was significantly delayed in Hif1α-/- (n=21) and Vhl-/- mice (n=19), while Hif2α-/- mice (n=18) had similar tumor progression compared to wildtype controls (n=19-20). Despite the delay in tumor growth, Hif1α-/- mice had greater total tumor weight (71%, p<0.01) and Vhl-/- mice had reduced total tumor weight (31%, p<0.05) on average compared to wildtype mice, with no difference in Hif2α-/- mice. Surprisingly, histological inspection of lung sections revealed that Hif1α-/- mice had greater incidence of macroscopic tumor nodules (n=15/21 Hif1α-/-, n=6/19 Hif1αf/f, p<0.05), and greater average lesion number and area (1.9- and 2.2-fold, respectively, p<0.05), but there was no difference in Ki67 staining in the lung nodules. In contrast, bone dissemination was significantly reduced in Hif1α-/- (56%, p<0.05) and Hif2α-/- mice (43%, p<0.01) and significantly increased in Vhl-/- mice (238%, p<0.01) as assessed by flow cytometry. However, there was no change in the expression of an 89-gene panel of metastasis associated genes or markers of EMT (Cdh1/2, Egfr, Notch1, Snai1-3, Twist1, Zeb1/2) or autophagy (Atg7, Becn1, Map1lc3a/b, Sqstm1, Ulk1) in Hif1α-/- primary tumors. None of the three transgenic strains showed a difference in dissemination to liver or brain. These data indicate that while both HIF1α and HIF2α drive tumor dissemination to bone, HIF1α is also essential for preventing metastatic outgrowth in the lung. It is therefore likely that the increase in bone dissemination when mammary fat pad VHL is deleted is due to elevated HIF1α and HIF2α signaling. Taken together, these data indicate a critical, yet divergent, role of HIF signaling in dissemination to bone and lung. Thus, targeted inhibition of HIF1α or HIF2α could be beneficial in preventing bone metastasis but may promote the outgrowth of lung-disseminated breast cancer cells. Citation Format: Vera M. Todd, Lawrence A. Vecchi, Katherine P. Snow, Lauren Himmel, Marjan Rafat, Rachelle W. Johnson. HIF signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner [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 2878.