Amanda Ernlund

mTORC1 and 2 coordinate transcriptional and translational reprogramming in resistance to DNA damage and replicative stress in breast cancer cells.

ABSTRACT mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. mTOR exists in two complexes: mTORC1, which stimulates protein, lipid, and ribosome biosynthesis, and mTORC2, which regulates cytoskeleton functions. While mTOR is known to be involved in the DNA damage response, little is actually known regarding the functions of mTORC1 compared to mTORC2 in this regard or the respective impacts on transcriptional versus translational regulation. We show that mTORC1 and mTORC2 are both required to enact DNA damage repair and cell survival, resulting in increased cancer cell survival during DNA damage. Together mTORC1 and -2 enact coordinated transcription and translation of protective cell cycle and DNA replication, recombination, and repair genes. This coordinated transcriptional-translational response to DNA damage was not impaired by rapalog inhibition of mTORC1 or independent inhibition of mTORC1 or mTORC2 but was blocked by inhibition of mTORC1/2. Only mTORC1/2 inhibition reversed cancer cell resistance to DNA damage and replicative stress and increased tumor cell killing and tumor control by DNA damage therapies in animal models. When combined with DNA damage, inhibition of mTORC1/2 blocked transcriptional induction more strongly than translation of DNA replication, survival, and DNA damage response mRNAs.

Hyperactive mTOR and MNK1 phosphorylation of eIF4E confer tamoxifen resistance and estrogen independence through selective mRNA translation reprogramming

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming.

A genomic ruler to assess oncogenic transition between breast tumor and stroma

Background Cancers induce gene expression alterations in stroma surrounding tumors that supports cancer progression. However, it is actually not at all known the extent of altered stromal gene expression enacted by tumors nor the extent to which altered stromal gene expression penetrates the stromal tissue. Presently, post-surgical “tumor-free” stromal tissue is determined to be cancer-free based on solely on morphological normality—a criteria that has not changed in more than 100 years despite the existence of sophisticated gene expression data to the contrary. We therefore investigated the extent to which breast tumors alter stromal gene expression in three dimensions in women undergoing mastectomy with the intent of providing a genomic determination for development of future risk of recurrence criteria, and to inform the need for adjuvant full-breast irradiation. Methods and findings Genome-wide gene expression changes were determined in histopathologically normal breast tissue in 33 women undergoing mastectomy for stage II and III primary invasive ductal carcinoma at serial distances in three dimensions from the tumor. Gene expression was determined by genome-wide mRNA analysis and subjected to metagene mRNA characterization. Tumor-like gene expression signatures in stroma were identified that surprisingly transitioned to a plastic, normalizing homeostatic signature with distance from tumor. Stroma closest to tumor displayed a pronounced tumor-like signature enriched in cancer-promoting pathways involved in disruption of basement membrane, cell migration and invasion, WNT signaling and angiogenesis. By 2 cm from tumor in all dimensions, stromal tissues were in transition, displaying homeostatic and tumor suppressing gene activity, while also expressing cancer supporting pathways. Conclusions The dynamics of gene expression in the post-tumor breast stroma likely co-determines disease outcome: reversion to normality or transition to transformation in morphologically normal tissue. Our stromal genomic signature may be important for personalizing surgical and adjuvant therapeutic decisions and risk of recurrence.

mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs

Platinum resistance is a major cause of treatment failure and mortality in epithelial ovarian cancer. mTORC1/2 inhibitors, which impair mRNA translation, can re-sensitize resistant ovarian cancer cells to platinum chemotherapy but the mechanism remains poorly described. Using platinum-resistant OVCAR-3 cells treated with the selective mTORC1/2 inhibitor INK128/MLN128, we conducted genome-wide transcription and translation studies and analyzed the effect on cell proliferation, AKT-mTOR signaling and cell survival, to determine whether carboplatin resistance involves selective mRNA translational reprogramming, and whether it is sensitive to mTORC1/2 inhibition. Gene ontology and Ingenuity Pathway Analysis (IPA) were used to categorize gene expression changes into experimentally authenticated biochemical and molecular networks. We show that carboplatin resistance involves increased mTORC1/2 signaling, resulting in selective translation of mRNAs involved in DNA damage and repair responses (DDR), cell cycle and anti-apoptosis (survival) pathways. Re-sensitization of ovarian cancer cell killing by carboplatin required only modest mTORC1/2 inhibition, with downregulation of protein synthesis by only 20-30%. Genome-wide transcriptomic and translatomic analyses in OVCAR-3 cells revealed that the modest downregulation of global protein synthesis by dual mTORC1/2 inhibition is associated with greater selective inhibition of DDR, cell cycle and survival mRNA translation, which was confirmed in platinum-resistant SKOV-3 cells. These data suggest a clinical path to re-sensitize platinum resistant ovarian cancer to platinum chemotherapy through partial inhibition of mTORC1/2, resulting in selective translation inhibition of DDR and anti-apoptosis protective mRNAs.

A widespread alternate form of cap-dependent mRNA translation initiation

Translation initiation of most mammalian mRNAs is mediated by a 5′ cap structure that binds eukaryotic initiation factor 4E (eIF4E). However, inactivation of eIF4E does not impair translation of many capped mRNAs, suggesting an unknown alternate mechanism may exist for cap-dependent but eIF4E-independent translation. We show that DAP5, an eIF4GI homolog that lacks eIF4E binding, utilizes eIF3d to facilitate cap-dependent translation of approximately 20% of mRNAs. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factors and receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair and translation initiation, among other mRNAs. Mass spectrometry and crosslinking studies demonstrate that eIF3d is a direct binding partner of DAP5. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E-independent capped-mRNA translation. These studies disclose a widespread and previously unknown mechanism for cap-dependent mRNA translation by DAP5-eIF3d complexes.Binding of eIF4E to the 5′ cap of mRNAs is a key early step in canonical translation initiation, but the requirement for eIF4E is not universal. Here the authors show that the eIF4G homolog DAP5 interacts with eIF3 to promote cap-dependent translation of a significant number of mRNA in an eIF4E-independent manner.

MTOR inhibition favors the differentiation of human in vitro-induced regulatory T cell through selective protein synthesis

The immunological composition of the primary cancer, metastatic sites, and stromal tissue determine cancer progression and treatment response. Breast cancers produce cytokines and chemokines that attract and polarize immune cells in a manner that promotes disease progression and metastasis. For example, macrophages are polarized to anti-inflammatory type II tumor activating macrophages (TAMs), and CD4+ T cells into tumor promoting, immune suppressing T regulatory cells (Tregs). n nStudies in mice and humans show that Tregs also are developed when inhibiting the kinase mTOR (mammalian target of rapamycin) via a poorly understood mechanism. mTOR forms two complexes in the cell, mTORC1 and mTORC2, which regulate multiple metabolic processes. In particular, mTORC1 is inhibited by the immunosuppressant rapamycin and stimulates the translation of mRNAs involved in cell growth and proliferation. Protein synthesis is a highly regulated process involving general mRNA and selective mRNA translation. Our hypothesis is that mTOR downregulation alters lymphocyte gene expression by favoring the translation of specific mRNAs required for Treg differentiation. n nWe generated human Tregs induced in culture (iTregs) by mTORC1 inhibition through RAD001 (everolimus), a rapalog used as an anti-cancer drug. By contrast, mTORC1/2 dual inhibitor PP242 blocked the growth of all the lymphocytes, pointing to a specific role of mTORC1 in Treg differentiation. In fact, we found that the proliferation of highly suppressive iTregs requires co-treatment with RAD001 and the cytokine TGFβ. Murine models do not require TGFβ, suggesting that caution should be used in extending findings from mouse to human Treg studies. Protein synthesis analysis in double-treated (RAD001+TGFβ), control-treated, RAD001- and TGFβ-treated cells shows that translation is greatly inhibited in the double-treated cells. Genome-wide translation profiling of mRNAs associated with actively translating ribosomes confirmed that only a selective pool of specific mRNAs is translated in the iTreg population. These specific mRNAs may be recruited to ribosome via an mTOR-independent mechanism involving PAIP2, and eIF4G adapter protein, and the ribosomal protein S25. n nOur work indicates the importance of selective translational regulation as an additional determinant of gene expression regulating T cell fate. We suggest that iTreg development is facilitated by selective translation of specific mRNAs whose recruitment to the ribosome is augmented when mTORC1 is inhibited. These findings also suggest that cancer treatments causing an improper balance of mTORC1 inhibition might attenuate the anti-tumor immune response through development of Tregs in the tumor microenvironment