CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.
Colorectal cancers (CRCs) that are sensitive to the anti-epidermal growth factor receptor (EGFR) antibodies cetuximab or panitumumab almost always develop resistance within several months of initiating therapy. We report the emergence of polyclonal KRAS, NRAS, and BRAF mutations in CRC cells with acquired resistance to EGFR blockade. Regardless of the genetic alterations, resistant cells consistently displayed mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) activation, which persisted after EGFR blockade. Inhibition of MEK1/2 alone failed to impair the growth of resistant cells in vitro and in vivo. An RNA interference screen demonstrated that suppression of EGFR, together with silencing of MEK1/2, was required to hamper the proliferation of resistant cells. Indeed, concomitant pharmacological blockade of MEK and EGFR induced prolonged ERK inhibition and severely impaired the growth of resistant tumor cells. Heterogeneous and concomitant mutations in KRAS and NRAS were also detected in plasma samples from patients who developed resistance to anti-EGFR antibodies. A mouse xenotransplant from a CRC patient who responded and subsequently relapsed upon EGFR therapy showed exquisite sensitivity to combinatorial treatment with MEK and EGFR inhibitors. Collectively, these results identify genetically distinct mechanisms that mediate secondary resistance to anti-EGFR therapies, all of which reactivate ERK signaling. These observations provide a rational strategy to overcome the multifaceted clonal heterogeneity that emerges when tumors are treated with targeted agents. We propose that MEK inhibitors, in combination with cetuximab or panitumumab, should be tested in CRC patients who become refractory to anti-EGFR therapies.
Understanding the role of single-nucleotide polymorphisms (SNPs) in the pathological process represents a unique experimental challenge especially when the variants occur outside of coding regions. The noncoding SNP rs61764370 located in the 3-untranslated region of Kirsten rat sarcoma viral oncogene homolog (KRAS) has been implicated as a risk factor for the development of cancer and the response to targeted therapies. This cancer-associated variant is thought to affect the binding of the microRNA let-7, which allegedly modulates KRAS expression. Using site-specific homologous recombination, we inserted the rs61764370:T>G KRAS gene variant in the colorectal cancer cell line SW48 (SW48 +SNP) and assessed the cellular and biochemical phenotype. We observed a significant increase in cellular proliferation, as well as a reduction in the levels of the microRNA let-7a, let-7b, and let-7c. Transcriptional and biochemical analysis showed no concomitant change in the KRAS protein expression or modulation of the downstream mitogen activated kinase or PI3K/AKT signaling. These results suggest that the cancer-associated rs61764370 variant exerts a biological effect not through transcriptional modulation of KRAS but rather by tuning the expression of the microRNA let-7.
MET, the high-affinity receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Tivantinib (ARQ197; Arqule), a staurosporine derivative that binds to the dephosphorylated MET kinase in vitro, is being tested clinically as a highly selective MET inhibitor. However, the mechanism of action of tivantinib is still unclear.
Cancer genomes display a complex blend of genetic lesions affecting oncogenes and tumor suppressor genes. Multiple modeling approaches indicate that 5-15 driver oncogenic events are required to achieve tumor progression in common epithelial cancers. In vitro, a lower number (2-3) of events is typically sufficient to achieve full transformation. We developed cellular models that closely resemble the occurrence of multiple genetic lesions to understand their role in tumor progression. Homologous recombination and transcriptional downregulation were used to recapitulate the co-occurrence of driver mutations targeting oncogenes and inactivation of tumor suppressor genes in human nontransformed epithelial cells. Knockdown of the tumor suppressor genes PTEN or RB1 was combined with mutagenic activation of individual oncogenes (EGFR, KRAS, BRAF, or PIK3CA), thus generating a combinatorial model. The simultaneous presence of oncogenic and tumor suppressive events resulted in distinct biochemical properties and anchorage-independent growth abilities. Notably, however, we found that even when up to four individual alterations were concomitantly present they were not sufficient to fully transform the target cells. Our results suggest that the close recapitulation of cancer lesions in not-transformed cells is essential to unveil their oncogenic potential and raise questions concerning the minimal requirements for neoplastic transformation of epithelial cells.
Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1s essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.
Patients with metastatic colorectal cancer who have KRAS codon 12- or KRAS codon 13-mutated tumors are presently excluded from treatment with the anti-epidermal growth factor receptor monoclonal antibody cetuximab.
Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.
The introduction of KRAS testing as a diagnostic tool to select patients for epidermal growth factor receptor (EGFR)-targeted cetuximab- or panitumumab-based therapies for metastatic colorectal cancer is widely regarded as a key advance in the field of personalized cancer medicine. Oncologists are now facing emerging issues in the treatment of metastatic colorectal cancer, including: (i) the identification of additional genetic determinants of primary resistance to EGFR-targeted therapy for further improving selection of patients; (ii) the explanation of rare cases of patients carrying KRAS-mutated tumors who have been reported to respond to either cetuximab or panitumumab and (iii) the discovery of mechanisms of secondary resistance to anti-EGFR antibody therapies. Here we discuss the potential role of comprehensive dissection of the key oncogenic nodes in the EGFR signaling cascade to predict resistance and sensitivity to EGFR monoclonal antibodies in metastatic colorectal cancer. Current data suggest that, together with KRAS mutations, the evaluation of BRAF and PIK3CA/PTEN alterations could also be useful for selecting patients with reduced chance to benefit from EGFR-targeted therapy. Furthermore, measuring EGFR gene copy number also appears relevant to positively identify responders. Up until now, each of these markers has been mainly assessed as a single event, often in retrospective analyses and patients series. As these molecular alterations display overlapping patterns of occurrence, this adds considerable complexity to the drawing of an algorithm suitable for clinical decision-making. We suggest that in the near future comprehensive molecular analysis of the entire oncogenic pathway triggered by the EGFR should be performed, thus enhancing the prediction ability of individual markers.
Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development. In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.
The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents an attractive target for therapeutic intervention. To investigate the primary phenotypes mediated by increased pathway signaling in a clean, patient-relevant context, an activating PIK3CA mutation (H1047R) was knocked-in to an endogenous allele of the MCF10A non-tumorigenic human breast epithelial cell line. Introduction of an endogenously mutated PIK3CA allele resulted in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype, compared to isogenic wild-type cells. The invasive phenotype was linked to enhanced PIP(3) production via a S6K-IRS positive feedback mechanism. Moreover, potent and selective inhibitors of PI3K were highly effective in reversing this phenotype, which is optimally revealed in 3-dimensional cell culture. In contrast, inhibition of Akt or mTOR exacerbated the invasive phenotype. Our results suggest that invasion is a core phenotype mediated by increased PTEN/PI3K pathway activity and that therapeutic agents targeting different nodes of the PI3K pathway may have dramatic differences in their ability to reverse or promote cancer metastasis.
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