Faculty Bibliography

The ovaries have a fixed pool of immature (primordial) follicles at birth known as the ovarian reserve. Activation or loss of follicles in this reserve causes an irreversible decline in reproductive function that culminates in menopause. Premenopausal women with cancer who are treated with conventional genotoxic chemotherapy have accelerated loss of the ovarian reserve, leading to subfertility and infertility. Cyclophosphamide (CY), a highly gonadotoxic drug, is widely used as part of combination cancer chemotherapy. This drug induces ovarian damage in large part by activating the mammalian/mechanistic target of rapamycin (mTOR) pathway, leading to activation of primordial follicles, follicular burnout, and premature menopause. As a result, the probability of pregnancy in premenopausal female cancer survivors is significantly diminished. There has been little progress in preserving ovarian function during cancer chemotherapy. As part of multiagent chemotherapeutic regimens, inhibitors of themTORC1 pathway have a growing role in cancer treatment and are being studied as treatment for a growing number of malignant and nonmalignant conditions. Mammalian/mechanistic target of rapamycin inhibitors block the primordial-to-primary follicle transition. The investigators used a clinically relevant mouse model of chemotherapy-induced gonadotoxicity to investigate whether inhibitors of mTOR could block CY-induced premature activation of primordial follicles and also preserve fertility during chemotherapy. Two inhibitors of the mTOR pathway were used: everolimus (RAD001), a drug clinically approved for treatment of tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer), and INK128, an experimental drug. Female mice were treated with CY weekly and then randomized to also receive either everolimus, INK128, or nothing.

Metastasis is the cause of 90% of cancer-related deaths. Selective mRNA translation has been found to be crucial for breast cancer development, progression and metastasis; however, there is a poor biological and mechanistic understanding of the role of translation in these processes. Our work and others have pioneered an understanding of translational regulation in breast cancer. We have previously shown that overexpression of initiation factor eIF4GI in advanced breast cancer cells selectively increases the translation of mRNAs encoding survival, cell cycle inhibition, and DNA damage and repair proteins, among other DNA damage-protective mRNAs. eIF4G consists of three family members: eIF4GI, eIF4GII, and the poorly studied eIF4G homolog, DAP5, the subject of this study. DAP5 lacks the N-terminal domain for eIF4E and PABP binding. DAP5 is therefore suspected to promote eIF4E-independent or IRES-driven translation of mRNAs involved in cell stress and oncogenesis, but its role in metastasis hasn't been investigated. We analyzed the NCI human tissue genome cancer atlas (TCGA) and found that higher DAP5 mRNA expression is strongly associated with estrogen receptor negative breast cancer metastasis with lower overall survival. We then engineered a highly metastatic breast cancer cell line MDA-MB-231TR to express dox-inducible shRNA to DAP5 or a non-silencing (NS) control. Tumors were developed in clinically relevant orthotopic mouse models, with and without DAP5 silencing by addition of dox to the drinking water. Remarkably, reduction of DAP5 expression by ~70% had no impact on primary tumor growth but fully eliminated metastasis to the lung. Genome-wide transcriptome and translatome analysis of DAP5 and in vivo ultraviolet-crosslinking and high-throughput sequencing (HITS-CLIP) identified specific mRNAs that interact directly with DAP5. A significant fraction of DAP5 mRNA targets are involved in cell death and survival, cell proliferation, cell mobility, DNA repair and translation initiation and do not have IRESs, suggesting a novel role for DAP5 in a non-classical translation initiation process upon cellular stress and metastasis. Our results suggest that the translation initiation factor DAP5 likely plays a critical role in breast cancer metastasis by a unique mechanism for metastasis-specific translation initiation and provides new concepts for therapeutic strategies involving translational regulation

Radiotherapy is under investigation for its ability to enhance responses to immunotherapy. However, the mechanisms by which radiation induces anti-tumour T cells remain unclear. We show that the DNA exonuclease Trex1 is induced by radiation doses above 12-18 Gy in different cancer cells, and attenuates their immunogenicity by degrading DNA that accumulates in the cytosol upon radiation. Cytosolic DNA stimulates secretion of interferon-beta by cancer cells following activation of the DNA sensor cGAS and its downstream effector STING. Repeated irradiation at doses that do not induce Trex1 amplifies interferon-beta production, resulting in recruitment and activation of Batf3-dependent dendritic cells. This effect is essential for priming of CD8+ T cells that mediate systemic tumour rejection (abscopal effect) in the context of immune checkpoint blockade. Thus, Trex1 is an upstream regulator of radiation-driven anti-tumour immunity. Trex1 induction may guide the selection of radiation dose and fractionation in patients treated with immunotherapy.

Equol, one of the most studied metabolites of the soy isoflavone daidzein, is associated with both anticancer and pro-cancer effects in breast cancer. These contradictory results have been shown to be concentration dependent, where high equol concentrations reduced non-metastatic breast cancer cell growth and proliferation, and reduced breast cancer risk. However, we have shown that at low physiological dietary concentrations, equol increases cell proliferation, tumor growth and metastasis in metastatic breast cancer cell models. Also, equol increases the expression of the eukaryotic initiation factor 4G (eIF4G), increasing the cap-independent protein synthesis of pro-cancer molecules (de La Parra et al., J Biol Chem. 2012, 287(50):41640-50 and J Biol Chem. 2015, 290(10):6047-57). To elucidate the role of equol in the regulation of protein synthesis in metastatic breast cancer cells, a polysome profile assay using an Affymetrix microarray was conducted in cells treated with the following experimental conditions: vehicle control, equol, eIF4G knockdown (kd), or both equol + eIF4G kd. The following equation was used to identify the mRNAs that were actively translated for each experimental condition: [(mRNA associated with heavy or light polysome fraction/total mRNA )/( control mRNA associated with heavy or light polysome fraction/total control mRNA)]. This analysis using a false discovery rate (FDR) of <0.05 demonstrated that >1000 protein coding and non-coding RNAs were differentially associated with polysomes in response to equol or eIF4G kd, but not for the combined treatment of equol + eIF4G kd. Ingenuity Pathway Analysis for mRNAs with a log² fold change >2, demonstrated significant changes in mRNAs of cancer invasion proteins like MMP1, chromatin remodeling proteins like Histones, and cancer-associated microRNAs (miRNAs) like miR21 and miR590. Therefore, our data has identified critical targets of equol involved in breast cancer metastasis regulation that are differentially expressed via translational control

The ovary contains oocytes within immature (primordial) follicles that are fixed in number at birth. Activation of follicles within this fixed pool causes an irreversible decline in reproductive capacity, known as the ovarian reserve, until menopause. Premenopausal women undergoing commonly used genotoxic (DNA-damaging) chemotherapy experience an accelerated loss of the ovarian reserve, leading to subfertility and infertility. Therefore, there is considerable interest but little effective progress in preserving ovarian function during chemotherapy. Here we show that blocking the kinase mammalian/mechanistic target of rapamycin (mTOR) with clinically available small-molecule inhibitors preserves ovarian function and fertility during chemotherapy. Using a clinically relevant mouse model of chemotherapy-induced gonadotoxicity by cyclophosphamide, and inhibition of mTOR complex 1 (mTORC1) with the clinically approved drug everolimus (RAD001) or inhibition of mTORC1/2 with the experimental drug INK128, we show that mTOR inhibition preserves the ovarian reserve, primordial follicle counts, serum anti-Mullerian hormone levels (a rigorous measure of the ovarian reserve), and fertility. Chemotherapy-treated animals had significantly fewer offspring compared with all other treatment groups, whereas cotreatment with mTOR inhibitors preserved normal fertility. Inhibition of mTORC1 or mTORC1/2 within ovaries was achieved during chemotherapy cotreatment, concomitant with preservation of primordial follicle counts. Importantly, our findings indicate that as little as a two- to fourfold reduction in mTOR activity preserves ovarian function and normal birth numbers. As everolimus is approved for tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer, these findings represent a potentially effective and readily accessible pharmacologic approach to fertility preservation during conventional chemotherapy.

MTOR COORDINATES GROWTH SIGNALS WITH METABOLIC PATHWAYS AND PROTEIN SYNTHESIS, AND IS HYPERACTIVATED IN MANY HUMAN CANCERS MTOR EXISTS IN TWO COMPLEXES, MTORC1 THAT STIMULATES PROTEIN, LIPID AND RIBOSOME BIOSYNTHESIS, AND MTORC2 THAT 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 IMPACT 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, 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, INCREASED TUMOR CELL KILLING AND TUMOR CONTROL BY DNA DAMAGE THERAPIES IN ANIMAL MODELS WHEN COMBINED WITH DNA DAMAGE, INHIBITION OF MTORC1/2 MORE STRONGLY BLOCKED TRANSCRIPTIONAL INDUCTION THAN TRANSLATION OF DNA REPLICATION, SURVIVAL, AND DNA DAMAGE RESPONSE MRNAS.

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10^th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

SLBP (stem-loop binding protein) is a highly conserved factor necessary for the processing, translation, and degradation of H2AFX and canonical histone mRNAs. We identified the F-box protein cyclin F, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the G2 ubiquitin ligase for SLBP. SLBP interacts with cyclin F via an atypical CY motif, and mutation of this motif prevents SLBP degradation in G2. Expression of an SLBP stable mutant results in increased loading of H2AFX mRNA onto polyribosomes, resulting in increased expression of H2A.X (encoded by H2AFX). Upon genotoxic stress in G2, high levels of H2A.X lead to persistent gammaH2A.X signaling, high levels of H2A.X phosphorylated on Tyr142, high levels of p53, and induction of apoptosis. We propose that cyclin F co-evolved with the appearance of stem-loops in vertebrate H2AFX mRNA to mediate SLBP degradation, thereby limiting H2A.X synthesis and cell death upon genotoxic stress.