Spectrum of somatic mutational features of colorectal tumors in ancestrally diverse populations.

Ancestrally diverse and admixed populations, including the Hispanic/Latino/a/x/e community, are underrepresented in cancer genetic and genomic studies. Leveraging the Latino Colorectal Cancer Consortium, we analyzed whole exome sequencing data on tumor/normal pairs from 718 individuals with colorectal cancer (128 Latino, 469 non-Latino) to map somatic mutational features by ethnicity and genetic ancestry. Global proportions of African, East Asian, European, and Native American ancestries were estimated using ADMIXTURE. Associations between global genetic ancestry and somatic mutational features across genes were examined using logistic regression. TP53 , APC , and KRAS were the most recurrently mutated genes. Compared to non-Latino individuals, tumors from Latino individuals had fewer KRAS (OR=0.64, 95%CI=0.41-0.97, p=0.037) and PIK3CA mutations (OR=0.55, 95%CI=0.31-0.98, p=0.043). Genetic ancestry was associated with presence of somatic mutations in 39 genes (FDR-adjusted LRT p<0.05). Among these genes, a 10% increase in African ancestry was associated with significantly higher odds of mutation in KNCN (OR=1.34, 95%CI=1.09-1.66, p=5.74×10 -3 ) and TMEM184B (OR=1.53, 95%CI=1.10-2.12, p=0.011). Among RMGs, we found evidence of association between genetic ancestry and mutation status in CDC27 (LRT p=0.0084) and between SMAD2 mutation status and AFR ancestry (OR=1.14, 95%CI=1.00-1.30, p=0.046). Ancestry was not associated with tumor mutational burden. Individuals with above-average Native American ancestry had a lower frequency of microsatellite instable (MSI-H) vs microsatellite stable tumors (OR=0.45, 95%CI=0.21-0.99, p=0.048). Our findings provide new knowledge about the relationship between ancestral haplotypes and somatic mutational profiles that may be useful in developing precision medicine approaches and provide additional insight into genomic contributions to cancer disparities. Significance: Our data in ancestrally diverse populations adds essential information to characterize mutational features in the colorectal cancer genome. These results will help enhance equity in the development of precision medicine strategies.

Correction: Transforming growth factor ß-induced epithelial-to-mesenchymal signature predicts metastasis-free survival in non-small cell lung cancer.

[This corrects the article DOI: 10.18632/oncotarget.26574.].

CDK4/6 inhibition synergizes with inhibition of P21-Activated Kinases (PAKs) in lung cancer cell lines.

Theoretically, small molecule CDK4/6 inhibitors (CDK4/6is) represent a logical therapeutic option in non-small cell lung cancers since most of these malignancies have wildtype RB, the key target of CDKs and master regulator of the cell cycle. Unfortunately, CDK4/6is are found to have limited clinical activity as single agents in non-small cell lung cancer. To address this problem and to identify effective CDK4/6i combinations, we screened a library of targeted agents for efficacy in four non-small cell lung cancer lines treated with CDK4/6 inhibitors Palbociclib or Abemaciclib. The pan-PAK (p21-activated kinase) inhibitor PF03758309 emerged as a promising candidate with viability ratios indicating synergy in all 4 cell lines and for both CDK4/6is. It is noteworthy that the PAKs are downstream effectors of small GTPases Rac1 and Cdc42 and are overexpressed in a wide variety of cancers. Individually the compounds primarily induced cell cycle arrest; however, the synergistic combination induced apoptosis, accounting for the synergy. Surprisingly, while the pan-PAK inhibitor PF03758309 synergizes with CDK4/6is, no synergy occurs with group I PAK inhibitors FRAX486 or FRAX597. Cell lines treated only with Ribociclib, FRAX486 or FRAX597 underwent G1/G0 arrest, whereas combination treatment with these compounds predominantly resulted in autophagy. Combining high concentrations of FRAX486, which weakly inhibits PAK4, and Ribociclib, mimics the autophagy and apoptotic effect of PF03758309 combined with Ribociclib. FRAX597, a PAKi that does not inhibit PAK4 did not reduce autophagy in combination with Ribociclib. Our results suggest that a unique combination of PAKs plays a crucial role in the synergy of PAK inhibitors with CDK4/6i. Targeting this unique PAK combination, could greatly improve the efficacy of CDK4/6i and broaden the spectrum of cancer treatment.

Characterization of epidermal growth factor receptor (EGFR) P848L, an unusual EGFR variant present in lung cancer patients, in a murine Ba/F3 model.

Lung cancer patients with mutations in epidermal growth factor receptor (EGFR) benefit from treatments targeting tyrosine kinase inhibitors (TKIs). However, both intrinsic and acquired resistance of tumors to TKIs are common, and EGFR variants have been identified that are resistant to multiple TKIs. In the present study, we characterized selected EGFR variants previously observed in lung cancer patients and expressed in a murine bone marrow pro-B Ba/F3 cell model. Among these EGFR variants, we report that an exon 20 deletion/insertion mutation S768insVGH is resistant to erlotinib (a first-generation TKI), but sensitive to osimertinib (a third-generation TKI). We also characterized a rare exon 21 germline variant, EGFR P848L, which transformed Ba/F3 cells and conferred resistance to multiple EGFR-targeting TKIs. Our analysis revealed that P848L (a) does not bind erlotinib; (b) is turned over less rapidly than L858R (a common tumor-derived EGFR mutation); (c) is not autophosphorylated at Tyr 1045 [the major docking site for Cbl proto-oncogene (c-Cbl) binding]; and (d) does not bind c-Cbl. Using viability assays including 300 clinically relevant targeted compounds, we observed that Ba/F3 cells transduced with EGFR P848L, S768insVGH, or L858R have very different drug-sensitivity profiles. In particular, EGFR P848L, but not L858R or S768insVGH, was sensitive to multiple Janus kinase 1/2 inhibitors. In contrast, cells driven by L858R, but not by P848L, were sensitive to multikinase MAPK/extracellular-signal-regulated kinase (ERK) kinase and ERK inhibitors including EGFR-specific TKIs. These observations suggest that continued investigation of rare TKI-resistant EGFR variants is warranted to identify optimal treatments for cancer.

Transforming growth factor ß-induced epithelial-to-mesenchymal signature predicts metastasis-free survival in non-small cell lung cancer.

Transforming growth factor beta (TGFß) plays a key role in regulating epithelial-to-mesenchymal transition (EMT). A gene expression signature (TGFß-EMT) associated with TGFß-induced EMT activities was developed using human Non-Small Cell Lung Carcinoma (NSCLC) cells treated with TGFß-1 and subjected to Affymetrix microarray analysis. The final 105-probeset TGFß-EMT signature covers 77 genes, and a NanoString assay utilized a subset of 60 of these genes (TGFß-EMTN signature). We found that the TGFß-EMT and TGFß-EMTN gene signatures predicted overall survival (OS) and metastasis-free survival (MFS). The TGFß-EMT signature was validated as prognostic of 5-year MFS in 3 cohorts: a 133 NSCLC tumor dataset (P = 0.0002), a NanoString assays of RNA isolated from formalin-fixed paraffin-embedded samples from these same tumors (P = 0.0015), and a previously published NSCLC MFS dataset (P = 0.0015). The separation between high and low metastasis signature scores was higher at 3 years (ΔMFS TGFß-EMT = -28.6%; ΔMFS TGFß-EMTN = -25.2%) than at 5 years (ΔMFS TGFß-EMT = -18.6%; ΔMFS TGFß-EMTN = -11.8%). In addition, the TGFß-EMT signature correlated with whether the cancer had already metastasized or not at time of surgery in a colon cancer cohort. The results show that the TGFß-EMT signature successfully discriminated lung cancer cell lines capable of undergoing EMT in response to TGFß-1 and predicts MFS in lung adenocarcinomas. Thus, the TGFß-EMT signature has the potential to be developed as a clinically relevant predictive biomarker, for example to identify those patients with resected early stage lung cancer who may benefit from adjuvant therapy.

Hyper-phosphorylation of Rb S249 together with CDK5R2/p39 overexpression are associated with impaired cell adhesion and epithelial-to-mesenchymal transition: Implications as a potential lung cancer grading and staging biomarker.

Prediction of lung cancer metastasis relies on post-resection assessment of tumor histology, which is a severe limitation since only a minority of lung cancer patients are diagnosed with resectable disease. Therefore, characterization of metastasis-predicting biomarkers in pre-resection small biopsy specimens is urgently needed. Here we report a biomarker consisting of the phosphorylation of the retinoblastoma protein (Rb) on serine 249 combined with elevated p39 expression. This biomarker correlates with epithelial-to-mesenchymal transition traits in non-small cell lung carcinoma (NSCLC) cells. Immunohistochemistry staining of NSCLC tumor microarrays showed that strong phospho-Rb S249 staining positively correlated with tumor grade specifically in the squamous cell carcinoma (SCC) subtype. Strong immunoreactivity for p39 positively correlated with tumor stage, lymph node invasion, and distant metastases, also in SCC. Linear regression analyses showed that the combined scoring for phospho-Rb S249, p39 and E-cadherin in SCC is even more accurate at predicting tumor staging, relative to each score individually. We propose that combined immunohistochemistry staining of NSCLC samples for Rb phosphorylation on S249, p39, and E-cadherin protein expression could aid in the assessment of tumor staging and metastatic potential when tested in small primary tumor biopsies. The intense staining for phospho-Rb S249 that we observed in high grade SCC could also aid in the precise sub-classification of poorly differentiated SCCs.

Somatic Mutations and Ancestry Markers in Hispanic Lung Cancer Patients.

INTRODUCTION: To address the lack of genomic data from Hispanic/Latino (H/L) patients with lung cancer, the Latino Lung Cancer Registry was established to collect patient data and biospecimens from H/L patients. METHODS: This retrospective observational study examined lung cancer tumor samples from 163 H/L patients, and tumor-derived DNA was subjected to targeted-exome sequencing (>1000 genes, including EGFR, KRAS, serine/threonine kinase 11 gene [STK11], and tumor protein p53 gene [TP53]) and ancestry analysis. Mutation frequencies in this H/L cohort were compared with those in a similar cohort of non-Hispanic white (NHW) patients and correlated with ancestry, sex, smoking status, and tumor histologic type. RESULTS: Of the adenocarcinomas in the H/L cohort (n = 120), 31% had EGFR mutations, versus 17% in the NHW control group (p < 0.001). KRAS (20% versus 38% [p = 0.002]) and STK11 (8% versus 16% [p = 0.065]) mutations occurred at lower frequency, and mutations in TP53 occurred at similar frequency (46% versus 40% [p = 0.355]) in H/L and NHW patients, respectively. Within the Hispanic cohort, ancestry influenced the rate of TP53 mutations (p = 0.009) and may have influenced the rate of EGFR, KRAS, and STK11 mutations. CONCLUSIONS: Driver mutations in H/L patients with lung adenocarcinoma differ in frequency from those in NHW patients associated with their indigenous American ancestry. The spectrum of driver mutations needs to be further assessed in the H/L population.

Phase I/II study of azacitidine, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer previously treated with docetaxel-based therapy.

INTRODUCTION: Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. PATIENTS AND METHODS: Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 + 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. RESULTS: In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6 with growth factor support. Prostate-specific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. CONCLUSION: The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients.

Transforming growth factor ß signaling overcomes dasatinib resistance in lung cancer.

Lung cancer is the second most common cancer and the leading cause of cancer-related deaths. Despite recent advances in the development of targeted therapies, patients with advanced disease remain incurable, mostly because metastatic non-small cell lung carcinomas (NSCLC) eventually become resistant to tyrosine kinase inhibitors (TKIs). Kinase inhibitors have the potential for target promiscuity because the kinase super family is the largest family of druggable genes that binds to a common substrate (ATP). As a result, TKIs often developed for a specific purpose have been found to act on other targets. Drug affinity chromatography has been used to show that dasatinib interacts with the TGFß type I receptor (TßR-I), a serine-threonine kinase. To determine the potential biological relevance of this association, we studied the combined effects of dasatinib and TGFß on lung cancer cell lines. We found that dasatinib treatment alone had very little effect; however, when NSCLC cell lines were treated with a combination of TGFß and dasatinib, apoptosis was induced. Combined TGFß-1 + dasatinib treatment had no effect on the activity of Smad2 or other non-canonical TGFß intracellular mediators. Interestingly, combined TGFß and dasatinib treatment resulted in a transient increase in p-Smad3 (seen after 3 hours). In addition, when NSCLC cells were treated with this combination, the pro-apoptotic protein BIM was up-regulated. Knockdown of the expression of Smad3 using Smad3 siRNA also resulted in a decrease in BIM protein, suggesting that TGFß-1 + dasatinib-induced apoptosis is mediated by Smad3 regulation of BIM. Dasatinib is only effective in killing EGFR mutant cells, which is shown in only 10% of NSCLCs. Therefore, the observation that wild-type EGFR lung cancers can be manipulated to render them sensitive to killing by dasatinib could have important implications for devising innovative and potentially more efficacious treatment strategies for this disease.

5-azacytidine reverses drug resistance in bladder cancer cells.

BACKGROUND: Patients with metastatic and muscle-invasive bladder cancer are commonly treated with cisplatin. A significant proportion of patients develop disease progression after an initial response to chemotherapy. Presently there is no standard of care for such patients. We examined whether pretreatment with an epigenetic agent would result in reversal of drug resistance. MATERIALS AND METHODS: Methylation of proapoptotic and cell cycle genes in bladder cancer cells was examined. Cisplatin- and docetaxel-resistant cells were generated. The effect of target of methylation-induced silencing (TMS1) expression and pretreatment of wild-type and drug-resistant cells with 5-azacytidine on chemosensitivity was determined. RESULTS: Unidirectional crossresistance of cisplatin-resistant UMUC3 cells to docetaxel was observed. Recombinant expression of TMS1 or pre-treatment of wild-type and drug-resistant cells with 5-azacytidine resulted in enhanced sensitivity to cisplatin and docetaxel. CONCLUSION: Our results indicate that epigenetic therapy may restore sensitivity to chemotherapeutic agents in bladder cancer cells.

Methylation-mediated silencing of TMS1 in pancreatic cancer and its potential contribution to chemosensitivity.

BACKGROUND: Resistance to chemotherapeutic agents, resulting in part from epigenetic silencing of pro-apoptotic genes, is one of the causes of treatment failure of pancreatic cancer. We examined whether epigenetic silencing of target of methylation induced silencing 1 (TMS1) contributes to resistance to chemotherapy in pancreatic cancer. MATERIALS AND METHODS: Methylation analysis was performed by methylation-specific PCR (MS-PCR) and gene expression was analyzed by quantitative reverse transcriptase PCR (qRT-PCR). MIA PaCa-2 cells were transfected with pCMV6-XL5/TMS1 plasmid and the effect of TMS1 expression on sensitivity to gemcitabine and docetaxel was determined. Cell viability was measured using Cell Titer Blue assay. RESULTS: TMS1 expression was repressed in MIA PaCa-2 cells by DNA methylation. Up-regulation of TMS1 by recombinant gene expression in MIA PaCa-2 cells or by pre-treatment of these cells with 5-azacytidine resulted in enhanced sensitivity to gemcitabine and docetaxel. CONCLUSION: Our results suggest that TMS1 is a potential therapeutic target in pancreatic cancer.

Serum free circulating DNA is a useful biomarker to distinguish benign versus malignant prostate disease.

BACKGROUND: Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a "representative" patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting. METHODS: The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene. RESULTS: Patients with PSA < or = 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA < or =180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA. CONCLUSIONS: Our results indicate that fcDNA may improve the specificity of prostate cancer screening. IMPACT: Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies.

Methylation mediated silencing of TMS1 in breast cancer and its potential contribution to docetaxel cytotoxicity.

BACKGROUND: The tumor suppressor gene TMS1 (target of methylation-induced silencing) has been described in the literature as a pro-apoptotic gene. This study examined the methylation status of TMS1 in breast cancer cells and its potential role in sensitivity to docetaxel chemotherapy. MATERIALS AND METHODS: Methylation of the TMS1 promoter was examined by methylation-specific PCR (MS-PCR) and gene expression was analyzed by reverse transcriptase PCR (RT-PCR). Apoptosis was evaluated by annexin V/propidium iodide staining followed by flow cytometric analysis. RESULTS AND CONCLUSION: The TMS1 promoter was unmethylated in ZR75-1, MB-231 and MCF7 cells which expressed the gene and partially methylated in SKBR3 and Hs578t cells in which TMS1 expression was down-regulated. Treatment of SKBR3 and Hs578t cells with demethylating agents resulted in reactivation of the TMS1 gene. Pretreatment with 5-azacytidine increased sensitivity to docetaxel treatment in SKBR3 and Hs578t cells, indicating that TMS1 reactivation in these cells may contribute to docetaxel sensitivity.

Methylation-mediated repression of GADD45alpha in prostate cancer and its role as a potential therapeutic target.

Defects in apoptotic pathway contribute to uncontrolled proliferation of cancer cells and confer resistance to chemotherapy. Growth arrest and DNA damage inducible, alpha (GADD45alpha) is up-regulated on docetaxel treatment and may contribute to docetaxel-mediated cytotoxicity. We examined the mechanism of regulation of GADD45alpha in prostate cancer cells and the effect of its up-regulation on sensitivity to docetaxel chemotherapy. Expression of GADD45alpha in PC3 cells was higher than that in Du145 and LNCaP cells (17- and 12-fold, respectively; P < 0.05). Although the proximal promoter region was unmethylated in all three cell lines, methylation of a 4 CpG region upstream of the proximal promoter correlated inversely with gene expression levels. Methylation was reversed by treatment of Du145 and LNCaP cells with DNA methyltransferase inhibitors, leading to reactivation of GADD45alpha expression in these cells. The 5' 4 CpG region was also frequently methylated in prostate cancer tissues. Methylation of this region correlated inversely with gene expression in prostate cancer and benign prostate tissues. The methyl binding protein MeCP2 was associated with the methylated 4 CpGs in Du145 cells, and knockdown of MeCP2 in these cells (Du145 MeCP2(-)) led to a significantly increased expression of GADD45alpha (3-fold; P = 0.035) without affecting the methylation status of the gene. Enhanced sensitivity to docetaxel was observed by up-regulation of GADD45alpha in Du145 cells by recombinant expression of GADD45alpha or pretreatment with 5-azacytidine. Our results show that GADD45alpha is epigenetically repressed and is a potential target for treatment of prostate cancer.

Methylation-mediated silencing of genes is not altered by selenium treatment of prostate cancer cells.

BACKGROUND: The role of selenium in reducing the risk of multiple cancers has been described in the literature. Although reports have described the antiproliferative and pro-apoptotic function of selenium by up-regulation of genes in these pathways, information is lacking on the target mechanisms of selenium on specific genes. This study examines whether selenium treatment alters the methylation status of epigenetically silenced genes in prostate cancer cells. MATERIALS AND METHODS: Methylation of glutathione sulfotransferase pi (GSTP1) and Ras associated family 1A (RASSF1A) genes was studied using methylation sensitive PCR (MS-PCR). Gene expression was studied using Reverse Transcriptase PCR and Western Blotting. RESULTS AND CONCLUSION: Treatment of prostate cancer cells with selenium did not alter the expression of genes that were silenced by DNA methylation. Furthermore, the methylation status of these genes remained unaltered after treatment with seleno-DL-methionine.