Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor–positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17β-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.
Todd W. Miller, Bryan T. Hennessy, Ana M. González-Angulo, Emily M. Fox, Gordon B. Mills, Heidi Chen, Catherine Higham, Carlos García-Echeverría, Yu Shyr, Carlos L. Arteaga
Several lines of evidence suggest that tumor cells show elevated activity of the NF-κB transcription factor, a phenomenon often resulting from constitutive activity of IκB kinase β (IKKβ). However, others have found that loss of NF-κB activity or IKKβ is tumor promoting. The role of NF-κB in tumor progression is therefore controversial and varies with tumor type. We sought to more extensively investigate the role IKKβ in melanoma tumor development by specifically disrupting Ikkb in melanocytes in an established mouse model of spontaneous melanoma, whereby HRasV12 is expressed in a melanocyte-specific, doxycycline-inducible manner in mice null for the gene encoding the tumor suppressor inhibitor cyclin-dependent kinase 4/alternative reading frame (Ink4a/Arf). Our results show that Ink4a/Arf–/– mice with melanocyte-specific deletion of Ikkb were protected from HRasV12-initiated melanoma only when p53 was expressed. This protection was accompanied by cell cycle arrest, with reduced cyclin-dependent kinase 2 (Cdk2), Cdk4, Aurora kinase A, and Aurora kinase B expression. Increased p53-mediated apoptosis was also observed, with decreased expression of the antiapoptotic proteins Bcl2 and survivin. Enhanced stabilization of p53 involved increased phosphorylation at Ser15 and reduced phosphorylation of double minute 2 (Mdm2) at Ser166. Together, our findings provide genetic and mechanistic evidence that mutant HRas initiation of tumorigenesis requires Ikkβ-mediated NF-κB activity.
Jinming Yang, Ryan Splittgerber, Fiona E. Yull, Sara Kantrow, Gregory D. Ayers, Michael Karin, Ann Richmond
Tumor cell resistance to ionizing radiation and chemotherapy is a major obstacle in cancer therapy. One factor contributing to this is integrin-mediated adhesion to ECM. The adapter protein particularly interesting new cysteine-histidine-rich 1 (PINCH1) is recruited to integrin adhesion sites and promotes cell survival, but the mechanisms underlying this effect are not well understood. Here we have shown that PINCH1 is expressed at elevated levels in human tumors of diverse origins relative to normal tissue. Furthermore, PINCH1 promoted cell survival upon treatment with ionizing radiation in vitro and in vivo by perpetuating Akt1 phosphorylation and activity. Mechanistically, PINCH1 was found to directly bind to protein phosphatase 1α (PP1α) — an Akt1-regulating protein — and inhibit PP1α activity, resulting in increased Akt1 phosphorylation and enhanced radioresistance. Thus, our data suggest that targeting signaling molecules such as PINCH1 that function downstream of focal adhesions (the complexes that mediate tumor cell adhesion to ECM) may overcome radio- and chemoresistance, providing new therapeutic approaches for cancer.
Iris Eke, Ulrike Koch, Stephanie Hehlgans, Veit Sandfort, Fabio Stanchi, Daniel Zips, Michael Baumann, Anna Shevchenko, Christian Pilarsky, Michael Haase, Gustavo B. Baretton, Véronique Calleja, Banafshé Larijani, Reinhard Fässler, Nils Cordes
Despite significant advancements in our understanding of cancer development, the molecular mechanisms that underlie the formation of liver cancer remain largely unknown. C/EBPα is a transcription factor that regulates liver quiescence. Phosphorylation of C/EBPα at serine 193 (S193-ph) is upregulated in older mice and is thought to contribute to age-associated liver dysfunction. Because development of liver tumors is associated with increasing age, we investigated the role of S193-ph in the development of liver cancer using knockin mice expressing a phospho-mimetic aspartic acid residue in place of serine at position 193 (S193D) of C/EBPα. The S193D isoform of C/EBPα was able to completely inhibit liver proliferation in vivo after partial hepatectomy. However, treatment of these mice with diethylnitrosamine/phenobarbital (DEN/PB), which induces formation of liver cancer, actually resulted in earlier development of liver tumors. DEN/PB treatment was associated with specific degradation of both the S193-ph and S193D isoforms of C/EBPα through activation of the ubiquitin-proteasome system (UPS). The mechanism of UPS-mediated elimination of C/EBPα during carcinogenesis involved elevated levels of gankyrin, a protein that was found to interact with the S193-ph isoform of C/EBPα and target it for UPS-mediated degradation. This study identifies a molecular mechanism that supports the development of liver cancer in older mice and potential therapeutic targets for the prevention of liver cancer.
Guo-Li Wang, Xiurong Shi, Simon Haefliger, Jingling Jin, Angela Major, Polina Iakova, Milton Finegold, Nikolai A. Timchenko
NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with α-galactosylceramide, the prototypic NKT cell–activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell–specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-γ. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell–targeted therapy in humans.
Hiroshi Watarai, Shin-ichiro Fujii, Daisuke Yamada, Andrei Rybouchkin, Sakura Sakata, Yuko Nagata, Midori Iida-Kobayashi, Etsuko Sekine-Kondo, Kanako Shimizu, Yohei Shozaki, Jafar Sharif, Masashi Matsuda, Shinobu Mochiduki, Takanori Hasegawa, Genta Kitahara, Takaho A. Endo, Tetsuro Toyoda, Osamu Ohara, Ken-ichi Harigaya, Haruhiko Koseki, Masaru Taniguchi
The gene encoding COMM domain–containing 1 (COMMD1) is a prototypical member of the COMMD gene family that has been shown to inhibit both NF-κB– and HIF-mediated gene expression. NF-κB and HIF are transcription factors that have been shown to play a role in promoting tumor growth, survival, and invasion. In this study, we demonstrate that COMMD1 expression is frequently suppressed in human cancer and that decreased COMMD1 expression correlates with a more invasive tumor phenotype. We found that direct repression of COMMD1 in human cell lines led to increased tumor invasion in a chick xenograft model, while increased COMMD1 expression in mouse melanoma cells led to decreased lung metastasis in a mouse model. Decreased COMMD1 expression also correlated with increased expression of genes known to promote cancer cell invasiveness, including direct targets of HIF. Mechanistically, our studies show that COMMD1 inhibits HIF-mediated gene expression by binding directly to the amino terminus of HIF-1α, preventing its dimerization with HIF-1β and subsequent DNA binding and transcriptional activation. Altogether, our findings demonstrate a role for COMMD1 in tumor invasion and provide a detailed mechanism of how this factor regulates the HIF pathway in cancer cells.
Bart van de Sluis, Xicheng Mao, Yali Zhai, Arjan J. Groot, Jeroen F. Vermeulen, Elsken van der Wall, Paul J. van Diest, Marten H. Hofker, Cisca Wijmenga, Leo W. Klomp, Kathleen R. Cho, Eric R. Fearon, Marc Vooijs, Ezra Burstein
Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.
Philipp Beckhove, Rolf Warta, Britt Lemke, Diana Stoycheva, Frank Momburg, Martina Schnölzer, Uwe Warnken, Hubertus Schmitz-Winnenthal, Rezvan Ahmadi, Gerhard Dyckhoff, Mariana Bucur, Simone Jünger, Thomas Schueler, Volker Lennerz, Thomas Woelfel, Andreas Unterberg, Christel Herold-Mende
Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein–like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell–derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.
Lizhi He, Alistair Ingram, Adrian P. Rybak, Damu Tang
Fanconi anemia (FA) is a rare human genetic disease caused by mutations in any one of 13 known genes that encode proteins functioning in one common signaling pathway, the FA pathway, or in unknown genes. One characteristic of FA is an extremely high incidence of cancer, indicating the importance of the FA pathway in tumor suppression. However, the role of this pathway in the development and progression of human cancers in individuals who do not have FA has not been clearly determined. Here, we report that elevated expression of what we believe to be a novel splice variant of FA complementation group L (FANCL), which we identified and named FAVL, can impair the FA pathway in non-FA human tumor cells and act as a tumor promoting factor. FAVL expression was elevated in half of the human carcinoma cell lines and carcinoma tissue samples tested. Expression of FAVL resulted in decreased FANCL expression by sequestering FANCL to the cytoplasm and enhancing its degradation. Importantly, this impairment of the FA pathway by FAVL elevation provided human cancer cells with a growth advantage, caused chromosomal instability in vitro, and promoted tumor development in a xenograft mouse model. These data indicate that FAVL impairment of the FA pathway likely contributes to the development of non-FA human cancers and therefore add a challenging layer of complexity to the pathogenesis of human cancer. We further believe that these data will prove useful for developing additional tools for fighting human cancer.
Jun Zhang, Deping Zhao, Hwan Ki Park, Hong Wang, Roy B. Dyer, Wanguo Liu, George G. Klee, Mark A. McNiven, Donald J. Tindall, Julian R. Molina, Peiwen Fei
Chronic stress is associated with hormonal changes that are known to affect multiple systems, including the immune and endocrine systems, but the effects of stress on cancer growth and progression are not fully understood. Here, we demonstrate that human ovarian cancer cells exposed to either norepinephrine or epinephrine exhibit lower levels of anoikis, the process by which cells enter apoptosis when separated from ECM and neighboring cells. In an orthotopic mouse model of human ovarian cancer, restraint stress and the associated increases in norepinephrine and epinephrine protected the tumor cells from anoikis and promoted their growth by activating focal adhesion kinase (FAK). These effects involved phosphorylation of FAKY397, which was itself associated with actin-dependent Src interaction with membrane-associated FAK. Importantly, in human ovarian cancer patients, behavioral states related to greater adrenergic activity were associated with higher levels of pFAKY397, which was in turn linked to substantially accelerated mortality. These data suggest that FAK modulation by stress hormones, especially norepinephrine and epinephrine, can contribute to tumor progression in patients with ovarian cancer and may point to potential new therapeutic targets for cancer management.
Anil K. Sood, Guillermo N. Armaiz-Pena, Jyotsnabaran Halder, Alpa M. Nick, Rebecca L. Stone, Wei Hu, Amy R. Carroll, Whitney A. Spannuth, Michael T. Deavers, Julie K. Allen, Liz Y. Han, Aparna A. Kamat, Mian M.K. Shahzad, Bradley W. McIntyre, Claudia M. Diaz-Montero, Nicholas B. Jennings, Yvonne G. Lin, William M. Merritt, Koen DeGeest, Pablo E. Vivas-Mejia, Gabriel Lopez-Berestein, Michael D. Schaller, Steven W. Cole, Susan K. Lutgendorf