Junan Li

Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4.

The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer.

P16(INK4A) (also known as P16 and MTS1), a protein consisting exclusively of four ankyrin repeats, is recognized as a tumor suppressor mainly because of the prevalence of genetic inactivation of the p16(INK4A) (or CDKN2A) gene in virtually all types of human cancers. However, it has also been shown that an elevated level of expression (upregulation) of P16 is involved in cellular senescence, aging, and cancer progression, indicating that the regulation of P16 is critical for its function. Here, we discuss the regulatory mechanisms of P16 function at the DNA level, the transcription level, and the posttranscriptional level, as well as their implications for the structure-function relationship of P16 and for human cancers.

Somatic INK4a‐ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

The INK4a‐ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor‐suppressor genes. The p16INK4a (p16) tumor‐suppressor gene product is a negative regulator of cyclin‐dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor‐suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0–10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single‐stranded conformational polymorphism (SSCP) analysis termed “cold” SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron‐based primers or a combination of intron‐ and exon‐based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin‐dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild‐type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29‐ to 38‐fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN. Mol. Carcinog. 30:26–36, 2001.

Frequent Alterations of p16INK4a and p14ARF in Oral Proliferative Verrucous Leukoplakia

Proliferative verrucous leukoplakia (PVL) represents a rare but highly aggressive form of oral leukoplakia with >70% progressing to malignancy. Yet, PVL remains biologically and genetically poorly understood. This study evaluated the cell cycle regulatory genes, p16INK4a and p14ARF, for homozygous deletion, loss of heterozygosity, and mutation events in 20 PVL cases. Deletion of exon 1β, 1α, or 2 was detected in 40%, 35%, and 0% of patients, respectively. Deletions of exons 1α and 1β markedly exceed levels reported in non-PVL dysplasias and approximate or exceed levels reported in oral squamous cell carcinomas. Allelic imbalance was assessed for markers reported to be highly polymorphic in squamous cell carcinomas and in oral dysplasias. Loss of heterozygosity was detected in 35.3%, 26.3%, and 45.5% of PVLs for the markers IFNα, D9S1748, and D9S171, respectively. INK4a and ARF sequence alterations were detected in 20% and 10% of PVL lesions, accordingly. These data show, for the first time, that both p16INK4a and p14ARF aberrations are common in oral verrucous leukoplakia; however, the mode and incidence of inactivation events differ considerably from those reported in non-PVL oral premalignancy. Specifically, concomitant loss of p16INK4a and p14ARF occurred in 45% of PVL patients greatly exceeding loss reported in non-PVL dysplastic oral epithelium (15%). In addition, p14ARF exon 1β deletions were highly elevated in PVLs compared with non-PVL dysplasias. These data illustrate that molecular alterations, even within a specific genetic region, are associated with distinct histologic types of oral premalignancy, which may affect disease progression, treatment strategies, and ultimately patient prognosis. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3179–87)

A structurally optimized celecoxib derivative inhibits human pancreatic cancer cell growth

Deregulation of the phosphatidylinositol 3-kinase (PI-3K)/PDK-l/Akt signaling cascade is associated with pancreatic cancer tumor invasion, angiogenesis, and tumor progression. As such, it has been postulated that PDK-1/Akt signaling inhibitors may hold promise as novel therapeutic agents for pancreatic cancer. Disadvantages of currently available Akt inhibitors include tumor resistance, poor specificity, potential toxicity, and poor bioavailability. Previous studies have demonstrated that OSU-03012, a celecoxib derivative, specifically inhibits PDK-1 mediated phosphorylation of Akt with IC50 values in the low mM range. Human pancreatic cancer cell lines AsPC-1, BxPC-3, Mia-PaCa 2, and PANC-1 were cultured in media containing varying concentrations of OSU-03012, 5-fluorouracil (5-FU), and gemcitabine, and changes in Akt phosphorylation and cell viability were evaluated using western blotting and a 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, respectively. Treatment with OSU-03012 resulted in decreased PDK-1-mediated Akt phosphorylation and cell growth inhibition for all cell lines with IC50 values ranging between 1.0 and 2.5 μM. Resistance to 5-FU and gemcitabine was observed in cell lines AsPC-1 and BxPC-3. Further analyses indicate that OSU-03012 induces both proapoptotic and antiproliferative effects in these cells. Taken together, these data suggest that OSU-03012 has potential value as a novel therapy for pancreatic cancer.

Results of an abbreviated phase-II study with the Akt Inhibitor MK-2206 in Patients with Advanced Biliary Cancer

Biliary cancers (BC) are rare, chemoresistant and are associated with a poor prognosis. Targeting the Akt pathway is of significance in BC. We hypothesized that the allosteric inhibitor MK-2206 will be active in BC. This was a multi-institutional phase II study of MK-2206 given to patients with advanced, refractory BC. The primary end point was overall response rate. We also characterized pharmacokinetic profiles of MK-2206 in these patients and explored its potential correlation with clinical outcomes. Eight patients were enrolled prior to early termination of the trial. All patients had received prior systemic therapy. The best response observed was stable disease, exceeding 12 weeks in two patients. Toxicities were mild and tolerable. MK-2206 exhibited a pharmacokinetic profile with an apparent slow absorption followed by biphasic elimination in these patients with BC. No significant association was observed between the pharmacokinetic properties of MK-2206 and clinical outcomes. MK-2206 as a single-agent in BC is tolerable with pharmacokinetic properties similar to patients with other solid tumors. No clinical activity was observed in this limited population. Further development of Akt inhibitors may need to focus on combinations with other molecular targeted agents, conventional cytotoxic chemotherapy and prospective patient selection.

Tumor suppressor p16INK4A/Cdkn2a alterations in 7, 12‐dimethylbenz(a)anthracene (DMBA)‐induced hamster cheek pouch tumors

The prevalence of p16INK4A/Cdkn2a genetic alterations in human oral cancers indicates that the p16 gene could be a potent and appropriate target for novel intervention. While chemically induced hamster cheek pouch (HCP) tumors are regarded as an appropriate surrogate model for human oral cancers because of their similarities to human oral cancers in both histology and genetics, little is known about the genetic events in the p16 gene in the HCP tumor model. The purpose of this study was to evaluate chemically induced HCP tumor specimens for potential inactivating p16 alterations. HCP tumors were induced with 7, 12‐dimethylbenz(a)anthracene (DMBA), and DNA extracted from 34 such specimens were analyzed for homozygous/hemizygous deletions, aberrant methylation of 5′ CpG islands, and point mutations using real‐time multiplex PCR, methylation‐specific PCR, and direct sequencing/cold single strand conformation polymorphism (SSCP), respectively. Homozygous deletions, hemizygous deletions, aberrant methylation of 5′‐CpG islands, and point mutation were identified in 11, 4, 9, and 1 of 34 specimens, respectively. While the overall incidence of p16 alterations was 70.6% (24 of 34 specimens), the majority of inactivating events (67.6%) stemmed from deletion or methylation, which is consistent with the observations found in human oral SCCs. Our results show the resemblance between chemically induced HCP tumors and their human counterparts in p16 genetic alterations, and strongly support the use of DMBA‐induced HCP tumor model in evaluating novel p16‐targeted therapy and prevention of human oral SCCs.

Neutral glycolipid composition of primary human brain tumors

Neutral glycolipids (NGL) were isolated and quantitated in 98 primary human brain tumors; 19 low grade astrocytomas (LGA), 12 anaplastic astrocytomas (AA), 37 high grade astrocytomas (HGA), 18 oligodendroglial tumors, and 12 primitive neuroectodermal tumors (PNET). In 38 of these, the nature of the hexose in the cerebroside was determined using immunothin-layer chromatographic techniques. Galactosylceramide (GalCer) was the major ceramide monohexoside (CMH), and glucosylcerebroside never comprised more than 6% of this fraction in any tumor type. Furthermore, there was no correlation between the proportion of glucosylcerebroside and histological diagnosis. AA had the most characteristic neutral glycolipid pattern, with high levels of total lipid, total neutral glycolipid, CMH, and ceramide dihexoside (CDH) but low water contents. Consistent with this glycolipid composition is the finding that AA usually had neither ceramide trihexoside (CTH) nor globoside. Oligodendrogliomas were somewhat similar to AA in having high levels of CMH and infrequently having CTH or globoside. However, oligodendrogliomas had low water and total lipid contents. PNET had low levels of total ipid, total NGL, and CMH, but frequently contained CTH and globoside. LGA had high water contents but low levels of total lipid and CMH. HGA tended to have intermediate levels of almost all constituents analyzed, probably reflecting the pronounced cellular heterogeneity of these tumors. The frequent presence of GalCer in astrocytomas raises the possibility that some of these contain a population of cells that are related to the oligodendroglial lineage. However, the low amounts of GalCer and infrequent presence of sulfatide in PNET is consistent with their lack of differentiation toward oligodendrocytes. It will be of interest to determine if the neutral glycolipid patterns reported here will correlate with patient survival and be of prognostic significance.

Contributions of conserved TPLH tetrapeptides to the conformational stability of ankyrin repeat proteins.

Ankyrin repeat (AR) proteins are one of the most abundant classes of repeat proteins and are involved in numerous physiological processes. These proteins are composed of various numbers of AR motifs stacked in a nearly linear fashion to adopt an elongated and nonglobular architecture. One salient feature prevalent in such a structural unit is the TPLH tetrapeptide or a close variant, T/SxxH, which initiates the helix-turn-helix conformation and presumably contributes to conformational stability through a hydrogen-bonding network. In the present study, we investigated the roles of T/SxxH motif in the stability, structure, and function of AR proteins by a systematic and rationalized mutagenic study on, followed by biochemical and biophysical characterization of, gankyrin, an oncogenic protein composed of seven ARs and six T/SxxH tetrapeptides, and P16, a tumor suppressor with four ARs but no TPLH tetrapeptide. Our results showed that this tetrapeptide is ineffectual on global structure and function, but contributes significantly to conformational stability when its stabilizing potentials are fully realized in the local conformation, including (1) the intra-AR hydrogen bonding involving the hydroxyl group; (2) the intra-AR and inter-AR hydrogen bonds involving the imidazole ring; and (3) the hydrophobic interaction associated with the Thr-methyl group. Considering that the capping and close-to-capping units tend to have more sequence diversity and more conformational variation, it could be also generally true that a T/SxxH motif close to the terminal repeats contributes little or even negatively to stability with respect to Ala substitution, but substantially stabilizes the global conformation when located in the middle of a long stretch of ARs.

Erlotinib in African Americans With Advanced Non–Small Cell Lung Cancer: A Prospective Randomized Study With Genetic and Pharmacokinetic Analyses

Prospective studies on epidermal growth factor receptor (EGFR) inhibitors in African Americans with non–small cell lung cancer (NSCLC) have not previously been performed. In this phase II randomized study, 55 African Americans with NSCLC received 150 mg/day erlotinib or a body weight–adjusted dose with subsequent escalations to the maximum‐allowable dose, 200 mg/day, to achieve rash. Erlotinib and OSI‐420 exposures were lower than those observed in previous studies, consistent with CYP3A pharmacogenetics implying higher metabolic activity. Tumor genetics showed only two EGFR mutations, EGFR amplification in 17/47 samples, eight KRAS mutations, and five EML4‐ALK translocations. Although absence of rash was associated with shorter time to progression (TTP), disease‐control rate, TTP, and 1‐year survival were not different between the two dose groups, indicating the dose‐to‐rash strategy failed to increase clinical benefit. Low incidence of toxicity and low erlotinib exposure suggest standardized and maximum‐allowable dosing may be suboptimal in African Americans.