The Ludwig Center at Harvard Medical School brings together investigators from across Harvard to develop strategies to overcome barriers that limit the efficacy of current and emerging cancer therapies.
Cancer cell survival is dependent on oxidative-stress defenses against reactive oxygen species (ROS) that accumulate during tumorigenesis. Here, we show a non-canonical oxidative-stress defense mechanism through TRPA1, a neuronal redox-sensing Ca2+-influx channel. In TRPA1-enriched breast and lung cancer spheroids, TRPA1 is critical for survival of inner cells that exhibit ROS accumulation. Moreover, TRPA1 promotes resistance to ROS-producing chemotherapies, and TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. TRPA1 does not affect redox status but upregulates Ca2+-dependent anti-apoptotic pathways. NRF2, an oxidant-defense transcription factor, directly controls TRPA1 expression, thus providing an orthogonal mechanism for protection against oxidative stress together with canonical ROS-neutralizing mechanisms. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies.
Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.
The PI3K-AKT kinase signaling pathway is frequently deregulated in human cancers, particularly breast cancer, where amplification and somatic mutations of PIK3CA occur with high frequency in patients. Numerous small-molecule inhibitors targeting both PI3K and AKT are under clinical evaluation, but dose-limiting toxicities and the emergence of resistance limit therapeutic efficacy. Various resistance mechanisms to PI3K inhibitors have been identified, including de novo mutations, feedback activation of AKT, or cross-talk pathways. We found a previously unknown resistance mechanism to PI3K pathway inhibition that results in AKT rebound activation. In a subset of triple-negative breast cancer cell lines, treatment with a PI3K inhibitor or depletion of PIK3CA expression ultimately promoted AKT reactivation in a manner dependent on the E3 ubiquitin ligase Skp2, the kinases IGF-1R (insulin-like growth factor 1 receptor) and PDK-1 (phosphoinositide-dependent kinase-1), and the cell growth and metabolism-regulating complex mTORC2 (mechanistic target of rapamycin complex 2), but was independent of PI3K activity or PIP3 production. Resistance to PI3K inhibitors correlated with the increased abundance of Skp2, ubiquitylation of AKT, cell proliferation in culture, and xenograft tumor growth in mice. These findings reveal a ubiquitin signaling feedback mechanism by which PI3K inhibitor resistance may emerge in aggressive breast cancer cells.
Triple negative breast cancers (TNBC) are genetically characterized by aberrations in TP53 and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole exome sequencing (WES) and RNAseq to identify somatic genetic alterations in mouse models of TNBCs driven by loss of Trp53 alone or in combination with Brca1. Amplifications or translocations that resulted in elevated oncoprotein expressions or oncoprotein-containing fusions, respectively, as well as frame-shift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. While the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNAseq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine guided TNBC treatment.
The precursor homocysteine is metabolized either through the methionine cycle to produce methionine or through the transsulfuration pathway to synthesize cysteine. Alternatively, cysteine can be obtained through uptake of its oxidized form, cystine. Many cancer cells exhibit methionine dependency such that their proliferation is impaired in growth media in which methionine is replaced by homocysteine. We showed that oncogenic PIK3CA and decreased expression of SLC7A11, a gene that encodes a cystine transporter also known as xCT, correlated with increased methionine dependency in breast cancer cells. Oncogenic PIK3CA was sufficient to confer methionine dependency to mammary epithelial cells, partly by decreasing cystine uptake through the transcriptional and posttranslational inhibition of xCT. Manipulation of xCT activity altered the proliferation of breast cancer cells in methionine-deficient, homocysteine-containing media, suggesting that it functionally contributed to methionine dependency. We propose that concurrent with decreased cystine uptake through xCT, PIK3CA mutant cells use homocysteine through the transsulfuration pathway to synthesize cysteine. Consequently, less homocysteine is available to produce methionine, contributing to methionine dependency. These results indicate that oncogenic PIK3CA alters methionine and cysteine utilization, partly by inhibiting xCT to contribute to the methionine dependency phenotype in breast cancer cells.
