We used a myeloid-specific Cre to conditionally delete CD82 in mouse osteoclasts and their precursors. In contrast to global loss of CD82 (gKO), conditional loss of CD82 (cKO) in osteoclasts does not affect cortical bone, osteoblasts, or adipocytes. CD82 loss results in greater trabecular volume and trabecular number but reduced trabecular space in 6-month old male mice. Though this trend is present in females it did not reach significance; whereas there was an increase in osteoclast numbers and eroded surface area only in female cKO mice. In vitro, there is an increase in osteoclast fusion and defects in actin assembly in both gKO and cKO mice, irrespective of sex. This is accompanied by altered osteoclast morphology and decreased release of CTX in vitro. Integrin αvβ3 expression is reduced, while integrin β1 is increased. Signaling to Src, Syk, and Vav are also compromised. We further discovered that expression of Clec2 and its ligand, Podoplanin, molecules that also signal to Syk and Vav, are increased in differentiated osteoclasts. Loss of CD82 reduces their expression. Thus, CD82 is required for correct assembly of the cytoskeleton and to limit osteoclast fusion, both needed for normal osteoclast function.

We previously identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.

We previously identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, this relationship was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Deregulated mTORC2 signaling in cancer is mostly associated with increased survival and metabolic reprogramming, but mTORC2 has also been linked to cancer cell migration, particularly in breast cancer. Interestingly, although Ras is rarely mutated in breast cancer, it is often upregulated due to amplification and overexpression of growth factor receptors. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that Ras and mTORC2 promote the migration of breast cancer cells, independently of the breast cancer molecular subtype. Using breast epithelial MCF10A cells transformed with HER2 or mutant Ras, we found that Ras promotes mTORC2 activation, and mTORC2-dependent migration and invasion. We further observed that while mutant Ras-transformed MCF10A cells display uncontrolled cell proliferation and an invasive phenotype, silencing of the mTORC2-dependent component Rictor mostly leads to loss of invasiveness. Together, our findings suggest that, whereas Ras activation of mTORC2 is likely to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays a key and general role in promoting the migration and invasion of breast cancer cells. Citation Format: Mollie E. Wiegand, Shannon E. Collins, Isabella N. Brown, Alyssa N. Werner, Isabelle M. Mundo, Ghassan Mouneimne, Pascale G. Charest. Ras-mediated activation of mTORC2 drives breast cancer cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2416.