Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

Summary We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

AFI score: Aberration Focality Index measures what proportion of the ARI score is distributed per potential target genes or other genetic elements spanned by the region. AFI is the ratio of a focality-weighted ARI to unweighted ARI (fwARI/ARI). As with ARI, AFI is calculated for each genomic position, separately for gain and loss samples. Focality weighting is performed with a conceptual model for the biological process of amplification and deletion that incorporates two fundamental aspects: (1) that CNA can progress in stage-wise fashion with progressive accumulation of extra copies associated with narrowing of the altered region, and (2) that DNA rearrangement within and across chromosomes may join nonadjacent sequence or delete intervening sequence such that a single amplicon may include non-contiguous genomic regions and be falsely represented as distinct CNAs in the aCGH profile. We consider three models for potential linkage of CNA across the profile: local, chromosomal and genomic. Local linkage treats each group of adjacent gained (or lost) segments as a contiguous discrete amplicon (or deletion) implying that the target genetic elements are spanned by the group of adjacent segments; Chromosome linkage considers that non-adjacent CNAs within the same chromosome represent a single amplicon (or deletion) with a shared set of targets. Genome linkage treats all CNA as if it belongs to a single complex amplicon (or deletion). Genome linkage is a conservative model, though not likely to be biologically accurate in most cases. Chromosomal linkage models the formation of amplicons with internal deletion, such as the co-amplification of CDK4 and MDM2 which typically excludes the adjoining region. Chromosomal linkage was used for the analysis in this study. Calculation of AFI is as follows: After all profiles have been analyzed, focality-weight ARI (fwARI) is calculated as for ARI, but using the fwMean S of each segment instead of mean log 2 ratio, mean S .

Peak selection and ROI bounding.
The dual indices ARI and AFI are determined for each point in the genome and can be used directly to select genomic regions enriched for gene targets of CNA. For the purpose of summarizing the distribution of these target-enriched regions, a heuristic algorithm was developed to select regions of interest (ROIs) bounding peaks in the product of the two indices: ARI x AFI, which is equivalent to fwARI. Local peaks in fwARI are analyzed and ROIs are bounded at falloff of 75% peak maximum, or at the minimum to the next peak, whichever is narrower. Each ROI is annotated by the mean ARI and AFI indices for the region, and sorted by the product of mean indices. ROIs are flagged if over half of the probes in the ROI lie within a region previously reported to be a copy number variation (CNV) in one of the 40 studies compiled for build hg17 in version 1 of the Database of Genomic Variants (http://projects.tcag.ca/variation/) (Iafrate et al., 2004).
Each core was examined and assigned a 0 to 4 score by an expert neuropathologist ( In addition, intensity of staining was scored as 0 to 3 for absent, weak, moderate and strong (Supplemental Table S4). Since normal brain cores were scored 0 or 1 for both proteins, cores with scores of 0 or 1 were assigned a "negative" call for expression. Staining intensities of all negative cores were considered weak or absent. While duplicate cores from the same tumor generally received the same call (positive or negative), in the case of p18 INK4C , cores from 4 cases were assigned different calls. Since basis for such this discordance cannot be determined and may be due to technical or biological variation (such as intratumoral heterogeneity), each core was considered an independent sample for this analysis. In parallel, these TMA sections were scanned by iCys laser scanning cytometer and analyzed to provide quantitative percentage of cells in each core that exhibited immunoreactivity.

Competitive PCR analysis of the genomic status of p16 INK4A and p18 INK4C
Competitive PCR was carried out for all exons of p16 INK4A and p18 INK4C to identify homozygous deletions. Between four and eight primer pairs were designed to each exon before being tested against a series of twenty control primer pairs. For each exon the combination that gave the best multiplex PCR result with products of equal intensity was then used to screen the core cell line set. Single exon deletions of ARF exon 1 and p16 INK4A exon 1 were confirmed using a different primer set in a non-multiplex assay. PCR conditions are listed in Methods and primer sequences are listed in Supplemental Table S6.   Table S4). 25% of the informative cores were negative for p18 INK4C protein expression and 67% of these also were negative for p16 INK4A protein expression.
Final calls for expression (positive or negative) were based on neuropathologist reading.
Parameters for the scores were as followed: