PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer.

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER + breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER + tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER + metastatic samples. PAK4 activity increases in ER + models of acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER + breast cancers.

Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio.

Previous reports indicate that mice deficient for cathepsin K (Ctsk), a key protease in osteoclastic bone resorption, develop osteopetrosis due to their inability to properly degrade organic bone matrix. Some features of the phenotype of Ctsk knockout mice, however, suggest the presence of mechanisms by which Ctsk-deficient mice compensate for the lack of cathepsin K. To study these mechanisms in detail, we generated Ctsk-deficient (Ctsk-/-) mice and analyzed them at the age of 2, 7, and 12 months using peripheral quantitative computed tomography, histomorphometry, resorption marker measurements, osteoclast and osteoblast differentiation cultures, and gene expression analyses. The present study verified the previously published osteopetrotic features of Ctsk-deficient mice. However, these changes did not exacerbate during aging indicating the absence of Ctsk to have its most severe effects during the rapid growth period. Resorption markers ICTP and CTX were decreased in the media of Ctsk-/- osteoclasts cultured on bone slices indicating impaired bone resorption. Ctsk-/- mice exhibited several mechanisms attempting to compensate for Ctsk deficiency. The number of osteoclasts in trabecular bone was significantly increased in Ctsk-/- mice compared to controls, as was the number of osteoclast precursors in bone marrow. The mRNA levels for receptor activator of nuclear factor (kappa)B ligand (RANKL) in Ctsk-/- bones were increased resulting in increased RANKL/OPG ratio favoring osteoclastogenesis. In addition, expression of mRNAs of osteoclastic enzymes (MMP-9, TRACP) and for osteoblastic proteases (MMP-13, MMP-14) were increased in Ctsk-/- mice compared to controls. Impaired osteoclastic bone resorption in Ctsk-/- mice results in activation of osteoblastic cells to produce increased amounts of other proteolytic enzymes and RANKL in vivo. We suggest that increased RANKL expression mediates enhanced osteoclastogenesis and increased protease expression by osteoclasts. These observations underline the important role of osteoblastic cells in regulation of osteoclast activity and bone turnover.

[Does tumor angiogenesis play a role in bone metastatic process?]. / Rôle de l'angiogenèse tumorale dans le processus métastatique osseux?

Angiogenesis plays an important role in the regulation of tumor growth and metastasis of numerous cancers. However, the role of angiogenesis in the bone metastasis process is totally unknown. Using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells in nude mice, we show that beside its well-know anti-angiogenic activity, angiostatin inhibits bone metastasis formation through a direct antiosteoclastic activity.

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity.

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.

Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice.

PURPOSE: Breast cancer frequently leads to incurable bone metastasis. Essential requirements for the development of bone metastasis are cell-cell and cell-matrix interactions, release of bioactive growth factors and cytokines, and removal of large amounts of bone matrix. Matrix metalloproteinases (MMPs) play an important role in all of these processes, but the possibility of using synthetic MMP inhibitors to decrease bone metastasis has received little attention. EXPERIMENTAL DESIGN: In the present study, we tested two general MMP inhibitors, BB-94 and GM6001, in a mouse model of breast cancer-induced bone metastasis. RESULTS: In a simulation of intervention therapy, mice were inoculated with breast cancer cells, and at the time of diagnosis of osteolytic lesions, the mice were treated for 10 or 15 consecutive days with BB-94 or GM6001, respectively. Both inhibitors reduced the growth of osteolytic lesions by >55% compared with control mice. Next, we simulated prevention therapy by initiating treatment with GM6001 at time of inoculation with cancer cells or 3 days earlier. Assessment of osteolytic lesions 28 days after inoculation showed that, in both cases, the treatment reduced the size of the osteolytic lesions by 60%, compared with that of control mice. Importantly, MMP inhibition also resulted in extension of symptom-free survival in the mice, whether the treatment was initiated at the time of diagnosis of osteolytic lesions or of cancer cell inoculation. CONCLUSIONS: The present study suggests the potential of synthetic MMP inhibitors as intervention or prevention treatments of breast cancer-induced osteolysis.