Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

Author Correction: Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion-Positive Intrahepatic Cholangiocarcinoma.

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983.

Liquid versus tissue biopsy for detecting acquired resistance and tumor heterogeneity in gastrointestinal cancers.

During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient1-3. Previous case reports and small case series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance4-8. However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.

Tyrosine phosphorylation controls nuclear localization and transcriptional activity of Ssdp1 in mammalian cells.

The LIM-HD proteins interact with different cofactors, including Ssdp1 to regulate development in a diverse range of species. The single stranded DNA binding protein (Ssdp1) is a member of an evolutionarily conserved family of proteins that regulate critical transcriptional processes during embryonic development. Ssdp1 is localized predominantly in the cytoplasm of 293T cells but is translocated to the nucleus when co-transfected with Lck, a member of the Src family of non-receptor tyrosine kinases. The Src tyrosine kinase inhibitor PP2 blocked the nuclear translocation of Ssdp1. Western blot analysis showed that co-expression of Ssdp1 and Lck in 293T cells induces Ssdp1 phosphorylation. Mutation of the Ssdp1 N terminal tyrosine residues 23 and 25 markedly reduced both the phosphorylation and the nuclear localization of Ssdp1. Lck enhanced the transcriptional activity of Ssdp1 in the context of known components of a LIM-homeodomain (LIM-HD)/cofactor complex. We propose that phosphorylation involving N-terminal tyrosine residues of Ssdp1 is a means of regulating its nuclear localization and subsequent transcriptional activation of LIM-HD complexes.

AKT1low Quiescent Cancer Cells Promote Solid Tumor Growth.

Human tumor growth depends on rapidly dividing cancer cells driving population expansion. Even advanced tumors, however, contain slowly proliferating cancer cells for reasons that remain unclear. Here, we selectively disrupt the ability of rapidly proliferating cancer cells to spawn AKT1low daughter cells that are rare, slowly proliferating, tumor-initiating, and chemotherapy-resistant, using ß1-integrin activation and the AKT1-E17K-mutant oncoprotein as experimental tools in vivo Surprisingly, we find that selective depletion of AKT1low slow proliferators actually reduces the growth of a molecularly diverse panel of human cancer cell xenograft models without globally altering cell proliferation or survival in vivo Moreover, we find that unusual cancer patients with AKT1-E17K-mutant solid tumors also fail to produce AKT1low quiescent cancer cells and that this correlates with significantly prolonged survival after adjuvant treatment compared with other patients. These findings support a model whereby human solid tumor growth depends on not only rapidly proliferating cancer cells but also on the continuous production of AKT1low slow proliferators. Mol Cancer Ther; 17(1); 254-63. ©2017 AACR.

Integrin ß1 activation induces an anti-melanoma host response.

TGF-ß is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-ß increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin ß1-mediated TGF-ß activation may also produce tumor suppression via an altered host response. In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin ß1-activation increases TGF-ß activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment. This is associated with attenuation of tumor growth and long-term survival benefit. Correspondingly, in human melanomas, TGF-ß1 correlates with integrin ß1/TGF-ß1 activation and the expression of markers for vasculature and stromal activation. Surprisingly, this integrin ß1/TGF-ß1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients. These correlations are unique to melanoma, in that we do not observe similar associations between ß1 integrin/TGF-ß1 activation and better long-term survival in other human tumor types. These results suggest that activation of TGF-ß1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study.

AKT Inhibition Promotes Nonautonomous Cancer Cell Survival.

Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication.

A mechanism for asymmetric cell division resulting in proliferative asynchronicity.

UNLABELLED: All cancers contain an admixture of rapidly and slowly proliferating cancer cells. This proliferative heterogeneity complicates the diagnosis and treatment of patients with cancer because slow proliferators are hard to eradicate, can be difficult to detect, and may cause disease relapse sometimes years after apparently curative treatment. While clonal selection theory explains the presence and evolution of rapid proliferators within cancer cell populations, the circumstances and molecular details of how slow proliferators are produced is not well understood. Here, a ß1-integrin/FAK/mTORC2/AKT1-associated signaling pathway is discovered that can be triggered for rapidly proliferating cancer cells to undergo asymmetric cell division and produce slowly proliferating AKT1(low) daughter cells. In addition, evidence indicates that the proliferative output of this signaling cascade involves a proteasome-dependent degradation process mediated by the E3 ubiquitin ligase TTC3. These findings reveal that proliferative heterogeneity within cancer cell populations, in part, is produced through a targetable signaling mechanism, with potential implications for understanding cancer progression, dormancy, and therapeutic resistance. IMPLICATIONS: These findings provide a deeper understanding of the proliferative heterogeneity that exists in the tumor environment and highlight the importance of designing future therapies against multiple proliferative contexts. VISUAL OVERVIEW: A proposed mechanism for producing slowly proliferating cancer cells. http://mcr.aacrjournals.org/content/early/2015/01/09/1541-7786.MCR-14-0474/F1.large.jpg.

Role of ldb1 in adult intestinal homeostasis.

Ldb1 is an essential co-regulator of transcription in embryonic development. It acts in conjunction with nuclear LIM-homeodomain and LIM-only proteins to control key events of organogenesis as precursor cells enter lineage specification. Here we ask whether Ldb1 exerts control over stem cell activation and differentiation throughout the life of the organism as required for tissue homeostasis. To help answer this question, we have generated conditional Ldb1 mouse mutants with an Ldb1 floxed/floxed;ROSA26CreER genotype. Tamoxifen treatment of 60 day-old mutant animals results in near-ubiquitous Cre-mediated Ldb1 inactivation. As a consequence, the stem cell microenvironment of intestinal crypts is drastically affected. Cells that normally express Ldb1 together with markers that identify them as lineage progenitors cease to retain bromodeoxyuridine and are gradually lost. Ldb1 inactivation in intestinal crypts and/or in neighboring mesenchymal cells also triggers activation of Wnt signaling in the stem cell niches of the small intestine. Cell proliferation is markedly increased in the epithelia of the small intestine, and Lgr5-expressing stem cells disappear from the base of the crypts. This perturbation of the normal process of tissue homeostasis causes apoptosis, and the animals do not survive. We conclude that Ldb1-mediated transcriptional regulation plays a major role in adult intestinal homeostasis.