To discover novel metastasis suppressor genes that are clinically relevant in common human cancers, we used isogenic human bladder cancer cell lines and used DNA microarray technology to identify genes whose expression diminishes as a function of invasive and metastatic competence. We then evaluated the expression profile of such genes in 105 pathologically characterized tumors from seven common organ sites, and we identified one gene, RhoGDI2, whose expression was diminished as a function of primary tumor stage and grade. When RhoGDI2 was transferred back into cells with metastatic ability that lacked its expression, it suppressed experimental lung metastasis but did not affect in vitro growth, colony formation, or in vivo tumorigenicity. In addition, RhoGDI2 reconstitution in these cells blocked invasion in an organotypic assay and led to a reduction of in vitro motility. These results indicate that RhoGDI2 is a metastasis suppressor gene, a marker of aggressive human cancer, and a promising target for therapy.
Abstract GDC-0941 is a class I PI3 kinase inhibitor of all 4 isoforms currently in phase II clinical trials in multiple indications, including ER+ breast cancer. The PI3 kinase pathway plays an important role in proliferation and survival and is known to be one of the most altered pathways in cancer. These alterations include PI3 kinase amplification, PI3 kinase mutations and PTEN loss, as well as numerous others. We have previously shown in-vitro and in-vivo data that alterations in this pathway can potentially help identify patients that are more likely to respond to a PI3 kinase inhibitor. A major outstanding question is mechanisms of intrinsic and acquired resistance to PI3K inhibitors. To address these questions we have selected multiple breast cancer cell lines harboring different pathway alterations (HER2, PIK3K mutations, PTEN loss) for resistance to PI3 kinase inhibition. Some of the resistant lines show cross resistance to inhibitors of mTOR and AKT (ie general pathway resistance), while others show specific resistance to PI3K or PI3K/mTOR inhibitors. We have characterized these resistant cell lines in detail at the molecular level and in terms of signaling pathway activation. Analyses performed include deep sequencing, reverse phase protein arrays, as well as focused Western blots and RTK arrays. We will discuss these results in detail and comment on candidate resistance mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5609. doi:1538-7445.AM2012-5609
<p>XLSX file - 602KB, Supplementary Table 5. Cancer cell lines analyzed in this study. Supplementary Table 6. Tumor tissues analyzed in this study. Supplementary Table 7. List of 340 well characterized hotspot mutations interrogated by the amplicon libraries in this study. Variants also assayed by asPCR are indicated. Supplementary Table 8. Variants detected in 66 cancer cell lines, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 9. Variants detected in 73 FFPE endometrial tumor tissues, after applying post-processing filters. Note that indels and predicted deleterious SNVs are reported for all interrogated genes and transcripts. Supplementary Table 10. For cell line data, concordance with COSMIC somatic mutation annotation.</p>
Abstract Purpose: Chronic myeloid leukemia (CML) can be effectively treated with BCR-ABL inhibitors such as imatinib, dasatinib, and nilotinib; however, resistance to these inhibitors develops over time causing patients to relapse. Recently, MEK inhibition was shown to synergize with BCR-ABL inhibitors to inhibit resistant CML cells harboring the T315I mutation (1). In this study we characterized mechanisms of resistance to BCR-ABL inhibitor in acquired resistant cells without BCR-ABL mutation by genomic and phosphoprotein profiling. We further evaluated the sensitivity of these resistant clones to MEK inhibitor, GDC0973, PI3K inhibitor, GDC0941, alone or in combination. Methods: The CML cell K562 was grown in an increasing concentration of imatinib or/and dasatinib to induce acquired resistance. Clones were isolated from the resistant pool and the GI50 of each clone was measured using the Cell-Titer Glo® viability assay. Genomic DNA from selected clones was tested using Oncoscan® (Affymetrix), which surveys whole genome for copy number variation (CNV) and 400 oncogenic mutations. Cell lysates were subjected to reverse phase protein array (RPPA) analysis of phosphorylation levels of ∼100 proteins. Genomic mutation and CNV profiles, as well as protein phosphorylation patterns of the clones were compared to the parental cells to identify potential pathways involved in resistance. Results: Four imatinib-resistant clones with varying GI50s (4–30 times higher than parental cells) were fully characterized with both Oncoscan and RPPA. None contained the T315I mutation. A genomic mutation scan showed that all the clones had multiple mutations affecting various signaling pathways including TGFβ (Smad-2), Wnt (APC), PI3K (PIK3CA) and EGFR. Likewise, whole genome copy number scan showed that CNV occurred in multiple genes including copy number gains in ADAM29, BMP2, Rspondin4 (Wnt) and PKA in multiple clones, and copy number losses in ADAMTS14, LIM-kinase and FzD9 (Wnt) in multiple clones. The protein phosphorylation patterns also showed multiple changes in the resistant clones. Three of the resistant clones (K14, 15 and 25) had 6-10-fold increased pMEK and pERK, relative to the parental cells. MEk inhibition resulted in a cytostatic response in the parental cells (GI50: 1900 nM), whereas resistant clones 14, 15, and 25 were highly sensitive to GDC0973-mediated apoptosis, with GI50s ranging from 60–75 nM. Clone 6 had lower pMEK and pERK levels than the others, but was still sensitive to GDC0973 (GI50: 600 nM). Phosphorylation of proteins in the PI3K pathway such as pS6, p70S6K and p-mTOR were elevated in all of the clones as compared to the parental cells (7–11 fold); however, none of these clones were sensitive to GDC0941 alone. Our data suggest that either GDC0973 or GDC0973 alone or in combination with GDC0941 could be effective at inducing cell death in resistant clones despite their various genetic backgrounds. Conclusion: The MEK inhibitor GDC0973 caused robust cellular apoptosis in all of the resistant clones in contrast to the cytostatic effect in parental cells. All of the clones had elevated PI3K signaling as shown by enhanced PI3K substrate phosphorylation. Despite the many genetic aberrations that occurred as cells became resistant; most noticeably changes in Wnt, PKA, ADAMs and BMP2 in all of the clones, resistant cells appear to channel escape/survival signals through the MAPK and/or PI3K pathway rendering them sensitive to GDC0973 alone or in combination with GDC0941.
The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non-small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, on-treatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted.
