Dai Horiuchi

Up-regulation of miR-21 by HER2/neu signaling promotes cell invasion

The cell surface receptor tyrosine kinase HER2/neu enhances tumor metastasis. Recent studies suggest that deregulated microRNA (miRNA) expression promotes invasion and metastasis of cancer cells; we therefore explored the possibility that HER2/neu signaling induces the expression of specific miRNAs involved in this process. We identified a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation and is functionally involved in HER2/neu-induced cell invasion. We show that miR-21 is up-regulated via the MAPK (ERK1/2) pathway upon stimulation of HER2/neu signaling in breast cancer cells, and overexpression of other ERK1/2 activators such as RASV12 or ID-1 is sufficient to induce miR-21 up-regulation in HER2/neu-negative breast cancer cells. Furthermore, the metastasis suppressor protein PDCD4 (programmed cell death 4) is down-regulated by miR-21 in breast cancer cells expressing HER2/neu. Our data reveal a mechanism for HER2/neu-induced cancer cell invasion via miRNA deregulation. In addition, our results identify miR-21 as a potential therapeutic target for the prevention of breast cancer invasion and metastasis.

Control of a Kinesin-Cargo Linkage Mechanism by JNK Pathway Kinases

Long-distance organelle transport toward axon terminals, critical for neuron development and function, is driven along microtubules by kinesins [1, 2]. The biophysics of force production by various kinesins is known in detail. However, the mechanisms of in vivo transport processes are poorly understood because little is known about how motor-cargo linkages are controlled. A c-Jun N-terminal kinase (JNK)-interacting protein (JIP1) has been identified previously as a linker between kinesin-1 and certain vesicle membrane proteins, such as Alzheimers APP protein and a reelin receptor ApoER2 [3, 4]. JIPs are also known to be scaffolding proteins for JNK pathway kinases [5, 6]. Here, we report evidence that a Drosophila ubiquitin-specific hydrolase and a JNK signaling pathway that it modulates can regulate a JIP1-kinesin linkage. The JNK pathway includes a MAPKKK (Wallenda/DLK), a MAPKK (Hemipterous/MKK7), and the Drosophila JNK homolog Basket. Genetic tests indicate that those kinases are required for normal axonal transport. Biochemical tests show that activation of Wallenda (DLK) and Hemipterous (MKK7) disrupts binding between kinesin-1 and APLIP1, which is the Drosophila JIP1 homolog. This suggests a control mechanism in which an activated JNK pathway influences axonal transport by functioning as a kinesin-cargo dissociation factor.

APLIP1, a Kinesin Binding JIP-1/JNK Scaffold Protein, Influences the Axonal Transport of Both Vesicles and Mitochondria in Drosophila

In a genetic screen for Kinesin heavy chain (Khc)-interacting proteins, we identified APLIP1, a neuronally expressed Drosophila homolog of JIP-1, a JNK scaffolding protein . JIP-1 and its homologs have been proposed to act as physical linkers between kinesin-1, which is a plus-end-directed microtubule motor, and certain anterograde vesicles in the axons of cultured neurons . Mutation of Aplip1 caused larval paralysis, axonal swellings, and reduced levels of both anterograde and retrograde vesicle transport, similar to the effects of kinesin-1 inhibition. In contrast, Aplip1 mutation caused a decrease only in retrograde transport of mitochondria, suggesting inhibition of the minus-end microtubule motor cytoplasmic dynein . Consistent with dynein defects, combining heterozygous mutations in Aplip1 and Dynein heavy chain (Dhc64C) generated synthetic axonal transport phenotypes. Thus, APLIP1 may be an important part of motor-cargo linkage complexes for both kinesin-1 and dynein. However, it is also worth considering that APLIP1 and its associated JNK signaling proteins could serve as an important signaling module for regulating transport by the two opposing motors.

Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation.

Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and nonphysiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a noncatalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and noninhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and nonredundant, rate-limiting roles in restriction point passage and S phase entry.

Adaptor protein Shc undergoes translocation and mediates up-regulation of the tyrosine kinase c-Src in EGF-stimulated A431 cells.

Shc is the adaptor protein that exists in three isoforms, P46, P52 and P66, and acts as a bridge between activated cell surface receptors and downstream signalling molecules which act in extracellular signal‐regulated cell events such as cell cycle progression. In our previous studies, Shc was shown to be a substrate of the tyrosine kinase c‐Src in vitro and in vivo.

Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways

A family of conserved serine/threonine kinases known as cyclin-dependent kinases (CDKs) drives orderly cell cycle progression in mammalian cells. Prior studies have suggested that CDK2 regulates S-phase entry and progression, and frequently shows increased activity in a wide spectrum of human tumors. Genetic KO/knockdown approaches, however, have suggested that lack of CDK2 protein does not prevent cellular proliferation, both during somatic development in mice as well as in human cancer cell lines. Here, we use an alternative, chemical-genetic approach to achieve specific inhibition of CDK2 kinase activity in cells. We directly compare small-molecule inhibition of CDK2 kinase activity with siRNA knockdown and show that small-molecule inhibition results in marked defects in proliferation of nontransformed cells, whereas siRNA knockdown does not, highlighting the differences between these two approaches. In addition, CDK2 inhibition drastically diminishes anchorage-independent growth of human cancer cells and cells transformed with various oncogenes. Our results establish that CDK2 activity is necessary for normal mammalian cell cycle progression and suggest that it might be a useful therapeutic target for treating cancer.

PIM1 kinase inhibition as a targeted therapy against triple-negative breast tumors with elevated MYC expression

Triple-negative breast cancer (TNBC), in which cells lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, is the breast cancer subtype with the poorest outcome. No targeted therapy is available against this subtype of cancer owing to a lack of validated molecular targets. We previously reported that signaling involving MYC—an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes—is disproportionally higher in triple-negative (TN) tumors than in receptor-positive (RP) tumors. Direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve. Here, by conducting a shRNA screen targeting the kinome, we identified PIM1, a non-essential serine–threonine kinase, in a synthetic lethal interaction with MYC. PIM1 expression was higher in TN tumors than in RP tumors and was associated with poor prognosis in patients with hormone- and HER2-negative tumors. Small-molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic mouse models of breast cancer by inhibiting the oncogenic transcriptional activity of MYC and restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that have elevated MYC expression.

Linking Tumor Mutations to Drug Responses via a Quantitative Chemical–Genetic Interaction Map

UNLABELLED There is an urgent need in oncology to link molecular aberrations in tumors with therapeutics that can be administered in a personalized fashion. One approach identifies synthetic-lethal genetic interactions or dependencies that cancer cells acquire in the presence of specific mutations. Using engineered isogenic cells, we generated a systematic and quantitative chemical-genetic interaction map that charts the influence of 51 aberrant cancer genes on 90 drug responses. The dataset strongly predicts drug responses found in cancer cell line collections, indicating that isogenic cells can model complex cellular contexts. Applying this dataset to triple-negative breast cancer, we report clinically actionable interactions with the MYC oncogene, including resistance to AKT-PI3K pathway inhibitors and an unexpected sensitivity to dasatinib through LYN inhibition in a synthetic lethal manner, providing new drug and biomarker pairs for clinical investigation. This scalable approach enables the prediction of drug responses from patient data and can accelerate the development of new genotype-directed therapies. SIGNIFICANCE Determining how the plethora of genomic abnormalities that exist within a given tumor cell affects drug responses remains a major challenge in oncology. Here, we develop a new mapping approach to connect cancer genotypes to drug responses using engineered isogenic cell lines and demonstrate how the resulting dataset can guide clinical interrogation.

Taking on challenging targets: making MYC druggable.

The transcription factor proto-oncogene c-MYC (hereafter MYC) was first identified more than 3 decades ago and has since been found deregulated in a wide variety of the most aggressive human malignancies. As a pleiotropic transcription factor, MYC directly or indirectly controls expression of hundreds of coding and noncoding genes, which affect cell cycle entry, proliferation, differentiation, metabolism, and death/survival decisions of normal and cancer cells. Tumors with elevated MYC expression often exhibit highly proliferative, aggressive phenotypes, and elevated MYC expression has been correlated with diminished disease-free survival for a variety of human cancers. The use of MYC overexpression or MYC-dependent transcriptional gene signatures as clinical biomarkers is currently being investigated. Furthermore, preclinical animal and cell-based model systems have been extensively utilized in an effort to uncover the mechanisms of MYC-dependent tumorigenesis and tumor maintenance. Despite our ever-growing understanding of MYC biology, currently no targeted therapeutic strategy is clinically available to treat tumors that have acquired elevated MYC expression. This article summarizes the progresses being made to discover and implement new therapies to kill MYC over-expressing tumors-a target that was once deemed undruggable.

Abstract C88: Genomics, advocacy, and emerging therapeutics to address triple-negative breast cancer (TNBC) outcome disparities.

Background: Collaborative team science provides a starting point for comprehensive change, and advocates have a unique and important role developing and engaging in transdisciplinary collaboratives that focus on new questions and new possibilities to advance the science of ethnic and medically underserved health care disparities. Participating in four areas : 1) research and programmatic support, 2) education and outreach, 3) policy and strategy, and 4) representation and advisory, the UCSF Breast Science Advocacy Core (BSAC) Program, a volunteer affiliate of the Breast Oncology Program (BOP), one of ten multidisciplinary research programs under the umbrella of the UCSF Helen Diller Comprehensive Cancer Center promotes a transformative, transdisciplinary, integrated environment to study the biological basis of the diseases that comprise breast cancer; to define the risk of developing or progressing with specific types of breast cancer; to develop novel interventions that work locally and globally to reduce morbidity and mortality from breast cancer and its treatment; and to leverage new collaborative research, education, and mentoring/training opportunities that address cancer outcome disparities. Advocates involved in KOMEN, DOD, PCORI, AND CBCRP funded research and training grants apply four core principles that forge synergy with NCI Advocacy Research Working Group Recommendations: 1)strategic innovation, 2)collaborative execution, 3)evidence based decision-making, and 4) ethical codes of conduct. Embracing transdisciplinary professionalism, researchers and advocates build on their track record as shared value partners committed to furthering the collective impact of science advocacy exchange (SAE). Study Objectives: Genomic analyses of patient tumors have unearthed an overwhelming number of recurrent somatic alterations in genes that have dramatic effects on tumor biology, patient drug responses, and clinical outcomes. In one study, high grade triple negative breast cancer (TNBC) accounts for 34% of breast cancers in African American women versus 21% in white women. A growing body of evidence has shown that African American women have biologically more aggressive disease, independent of social determinants, and suffer the highest mortality rates. While biological breakthroughs of the last decade have greatly advanced our understanding of cancer, in advanced TNBC, a poor prognosis subtype, there is an urgent need to translate this evolving patient genomic data into new therapeutic paradigms. Our study focuses on the intersection of synthetic lethal approaches, MYC driven human cancers, and immunotherapy as an “innovation agenda”. A distinct MYC vision highlights how overexpression is associated with aggressive outcomes and poor patient outcomes, and synthetic lethal strategies to target MYC (CDK inhibitors, PIM2, as well as the PDI immune pathways) have potential for addressing outcome disparities In African American Women with Triple Negative Breast Cancer (TNBC). Key Findings: We have developed a screening technique that can be used to rapidly and accurately identify potential synthetic lethal interactions in TNBC. This platform utilizes an isogenic cell line system that we have developed to model oncogene activation in TNBC. A growing body of evidence has shown that: 1) Quantitative approach maps genotype-specific drug responses in isogenic cells 2) Systematic discovery of biomarkers for cancer drugs under clinical investigation 3) Clinically actionable synthetic lethal interaction between MYC and dasatinib is discovered 4) Mechanism of dasatinib action through inhibition of LYN kinase is described Key Take-Away Message: The inclusion of advocates in convergent science settings remind academic stakeholders that research is there to benefit the patient as they attempt to spark innovation, democratize science, and support smarter interventions that expedite the incredible potential of future investments in bioscience within disparities arenas. Citation Format: Susan Samson, Alicia Y. Zhou, Maria Martins, Alexandra Corella, Dai Horiuchi, Christina Yau, Taha Rakshandehroo, John Gordan, Rebecca Levin, Jeff Johnson, John Jascur, Mike Shales, Antonio Sorrentino, Jaime Cheah, Paul Clemens, Alykhan Shamji, Stuart Schreiber, Nevan Krogan, Kevan Shokat, Frank McCormick, Sourav Bandyopadhyay, Andrei Goga. Genomics, advocacy, and emerging therapeutics to address triple-negative breast cancer (TNBC) outcome disparities. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C88.