Simultaneous screening of overexpressed genes in breast cancer for oncogenic drivers and tumor dependencies.

There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving ß-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment.

Challenges in Ras therapeutics in pancreatic cancer.

Pancreatic cancer is considered among the most aggressive and the least curable of all human malignancies. It is usually characterized by multiple aberrations in tumor suppressor genes and oncogenes, most notably activating mutations in KRAS. This review examines the various attempts that have been made to inhibit Kras and its downstream signaling pathways in pancreatic cancer with an emphasis on challenges related to clinical trials. Attempts include preventing the localization of Ras protein to the plasma membrane, inhibiting downstream oncogenic signaling by targeting Kras effectors such as MEK1/2, Erk1/2 or Akt singly or in combination, and directly inhibiting Kras protein. Most clinical trials have focused on inhibiting downstream effector pathways and clinical benefit has been limited due to compensatory mechanisms and toxicity associated with small therapeutic windows. Additionally, genetic screens have been conducted to identify gene or genes that could provide therapeutic vulnerabilities in mutant KRAS cells and provide a way to target mutant Kras protein only. We also discuss how potentially transforming clinical trials have failed in the past and what new strategies are on-going in clinical trials for pancreas cancer. For long-term success in targeting Kras, future efforts should focus on combinatorial strategies to more effectively block Kras pathways at multiple points, and improve translational application of pre-clinical data to the clinic.

Nuclear-cytoplasmic shuttling of Chibby controls beta-catenin signaling.

In the canonical Wnt pathway, beta-catenin acts as a key coactivator that stimulates target gene expression through interaction with Tcf/Lef transcription factors. Its nuclear accumulation is the hallmark of active Wnt signaling and is frequently associated with cancers. Chibby (Cby) is an evolutionarily conserved molecule that represses beta-catenin-dependent gene activation. Although Cby, in conjunction with 14-3-3 chaperones, controls beta-catenin distribution, its molecular nature remains largely unclear. Here, we provide compelling evidence that Cby harbors bona fide nuclear localization signal (NLS) and nuclear export signal (NES) motifs, and constitutively shuttles between the nucleus and cytoplasm. Efficient nuclear export of Cby requires a cooperative action of the intrinsic NES, 14-3-3, and the CRM1 nuclear export receptor. Notably, 14-3-3 docking provokes Cby binding to CRM1 while inhibiting its interaction with the nuclear import receptor importin-alpha, thereby promoting cytoplasmic compartmentalization of Cby at steady state. Importantly, the NLS- and NES-dependent shuttling of Cby modulates the dynamic intracellular localization of beta-catenin. In support of our model, short hairpin RNA-mediated knockdown of endogenous Cby results in nuclear accumulation of beta-catenin. Taken together, these findings unravel the molecular basis through which a combinatorial action of Cby and 14-3-3 proteins controls the dynamic nuclear-cytoplasmic trafficking of beta-catenin.

Chibby forms a homodimer through a heptad repeat of leucine residues in its C-terminal coiled-coil motif.

BACKGROUND: The Wnt/beta-catenin signaling pathway plays crucial roles in embryonic development and in maintenance of organs and tissues in adults. Chibby (Cby) is an evolutionarily conserved molecule that physically interacts with the key downstream coactivator beta-catenin and represses its transcriptional activation potential. Although Cby harbors a predicted coiled-coil motif in the C-terminal region, its molecular nature and functional importance remain largely unexplored. RESULTS: Here we report that Cby forms a stable complex with itself. Alanine substitutions of two or more of four critical leucine residues within the C-terminal heptad repeats completely eliminate the Cby-Cby interaction. The Cby oligomer predominantly exists as a homodimer. Furthermore, we found that dimerization-deficient Cby mutants still retain the ability to bind to beta-catenin and to repress beta-catenin-dependent gene activation. More importantly, Cby homodimerization is required for its efficient interaction with the nuclear import receptor importin-alpha and subsequent nuclear translocation. CONCLUSION: Our comprehensive mutational analysis of the Cby coiled-coil domain reveals that the four heptad leucine residues play an essential role in mediating Cby homodimerization. Although monomeric Cby is sufficient to bind to beta-catenin and block beta-catenin-mediated transcriptional activation, homodimer formation of Cby is indispensable for its efficient nuclear import.

Chibby cooperates with 14-3-3 to regulate beta-catenin subcellular distribution and signaling activity.

beta-Catenin functions in both cell-cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of beta-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although beta-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits beta-catenin-mediated transcriptional activation. Here, we identified 14-3-3epsilon and 14-3-3zeta as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3-binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with beta-catenin, causing beta-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of beta-catenin, thereby antagonizing beta-catenin signaling.

Chibby promotes adipocyte differentiation through inhibition of beta-catenin signaling.

The canonical Wnt/beta-catenin signaling pathway plays diverse roles in embryonic development and disease. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and in mice. Here, we report that the beta-catenin antagonist Chibby (Cby) is required for adipocyte differentiation. Cby is expressed in adipose tissue in mice, and Cby protein levels increase during adipogenic differentiation of 3T3-L1 cells. Ectopic expression of Cby induces spontaneous differentiation of these cells into mature adipocytes to an extent similar to that of dominant-negative Tcf-4. In contrast, depletion of Cby by RNA interference potently blocks adipogenesis of 3T3-L1 and mouse embryonic stem cells. In support of this, embryonic fibroblasts obtained from Cby-deficient embryos display attenuated differentiation to the adipogenic lineage. Mechanistically, Cby promotes adipocyte differentiation, in part by inhibiting beta-catenin, since gain or loss of function of Cby influences beta-catenin signaling in 3T3-L1 cells. Our results therefore establish Cby as a novel proadipogenic factor required for adipocyte differentiation.