Boning Gao

Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30–40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n = 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30–90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5′ region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.

Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations

We used CDK4/hTERT–immortalized normal human bronchial epithelial cells (HBEC) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, and MYC) and followed the stepwise transformation of HBECs to full malignancy. This model showed that: (i) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; (ii) genetically identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; (iii) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; (iv) high levels of KRASV12 are required for full malignant transformation of HBECs, however, prior loss of p53 function is required to prevent oncogene-induced senescence; (v) overexpression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; (vi) growth of parental HBECs in serum-containing medium induces differentiation, whereas growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); (vii) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; (viii) an mRNA signature derived by comparing tumorigenic versus nontumorigenic clones was predictive of outcome in patients with lung cancer. Collectively, our findings show that this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes. Visual Overview: http://mcr.aacrjournals.org/content/11/6/638/F1.large.jpg. Mol Cancer Res; 11(6); 638–50. ©2013 AACR. Visual Overview

Cerebellar Ataxia, Seizures, Premature Death, and Cardiac Abnormalities in Mice with Targeted Disruption of the Cacna2d2 Gene

CACNA2D2 is a putative tumor suppressor gene located in the human chromosome 3p21.3 region that shows frequent allelic imbalances in lung, breast, and other cancers. The alpha2delta-2 protein encoded by the gene is a regulatory subunit of voltage-dependent calcium channels and is expressed in brain, heart, and other tissues. Here we report that mice homozygous for targeted disruption of the Cacna2d2 gene exhibit growth retardation, reduced life span, ataxic gait with apoptosis of cerebellar granule cells followed by Purkinje cell depletion, enhanced susceptibility to seizures, and cardiac abnormalities. The Cacna2d2(tm1NCIF) null phenotype has much in common with that of Cacna1a mutants, such as cerebellar neuro-degeneration associated with ataxia, seizures, and premature death. A tendency to bradycardia and limited response of null mutants to isoflurane implicate alpha2delta-2 in sympathetic regulation of cardiac function. In summary, our findings provide genetic evidence that the alpha2delta-2 subunit serves in vivo as a component of P/Q-type calcium channels, is indispensable for the central nervous system function, and may be involved in hereditary cerebellar ataxias and epileptic disorders in humans.

CACNA2D2-mediated apoptosis in NSCLC cells is associated with alterations of the intracellular calcium signaling and disruption of mitochondria membrane integrity

The CACNA2D2 gene, a new subunit of the Ca2+-channel complex, was identified in the homozygous deletion region of chromosome 3p21.3 in human lung and breast cancers. Expression deficiency of the CACNA2D2 in cancer cells suggests a possible link of it to Ca2+ signaling in the pathogenesis of lung cancer and other cancers. We investigated the effects of overexpression of CACNA2D2 on intracellular Ca2+ contents, mitochondria homeostasis, cell proliferation, and apoptosis by adenoviral vector-mediated wild-type CACNA2D2 gene transfer in 3p21.3-deficient nonsmall cell lung cancer cell lines. Exogenous expression of CACNA2D2 significantly inhibited tumor cell growth compared with the controls. Overexpression of CACNA2D2 induced apoptosis in H1299 (12.5%), H358 (13.7%), H460 (22.3%), and A549 (50.1%) cell lines. Levels of intracellular free Ca2+ were elevated in AdCACNA2D2-transduced cells compared with the controls. Mitochondria membrane depolarization was observed prior to apoptosis in Ad-CACNA2D2 and Adp53-transduced H460 and A549 cells. Release of cyt c into the cytosol, caspase 3 activation, and PARP cleavage were also detected in these cells. Together, these results suggest that one of the pathways in CACNA2D2-induced apoptosis is mediated through disruption of mitochondria membrane integrity, the release of cyt c, and the activation of caspases, a process that is associated with regulation of cytosolic free Ca2+ contents.

The 3p21.3 Tumor Suppressor NPRL2 Plays an Important Role in Cisplatin-Induced Resistance in Human Non–Small-Cell Lung Cancer Cells

NPRL2 is one of the novel candidate tumor suppressor genes identified in the human chromosome 3p21.3 region. The NPRL2 has shown potent tumor suppression activity in vitro and in vivo and has been suggested to be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. In this study, we analyzed the endogenous expression of the NPRL2 protein and the cellular response to cisplatin in 40 non-small-cell lung cancer cell lines and found that expression of NPRL2 was significantly and reciprocally correlated to cisplatin sensitivity, with a Spearman correlation coefficient of -0.677 (P < 0.00001). Exogenously introduced expression of NPRL2 by N-[1-(2,3-dioleoyloxyl)propyl]-NNN-trimethylammoniummethyl sulfate:cholesterol nanoparticle-mediated gene transfer significantly resensitized the response to cisplatin, yielding a 40% greater inhibition of tumor cell viability and resulting in a 2- to 3-fold increase in induction of apoptosis by activation of multiple caspases in NPRL2-transfected cells compared with untransfected cells at an equal dose of cisplatin. Furthermore, a systemic treatment with a combination of NPRL2 nanoparticles and cisplatin in a human H322 lung cancer orthotopic mouse model significantly enhanced the therapeutic efficacy of cisplatin and overcame cisplatin-induced resistance (P < 0.005). These findings implicate the potential of NPRL2 as a biomarker for predicting cisplatin response in lung cancer patients and as a molecular therapeutic agent for enhancing response and resensitizing nonresponders to cisplatin treatment.

XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.

RASSF1A polymorphism A133S is associated with early onset breast cancer in BRCA1/2 mutation carriers

The tumor suppressor gene RASSF1A regulates cell cycle progression, apoptosis, and microtubule stability and is inactivated by promoter methylation in approximately 50% of breast cancers. It has been shown previously that the polymorphism A133S in RASSF1A reduces its ability to regulate cell cycle progression and this polymorphism is associated with an increased risk of breast cancer. We analyzed the frequency of RASSF1A A133S in 190 Caucasian women without breast cancer and 653 patients with breast cancer including 138 BRCA1 and BRCA2 (BRCA1/2) mutation carriers, 395 non-BRCA1/2 mutations carriers, and 120 untested for BRCA1/2 mutations. Patients with breast cancer had a higher frequency of A133S than the controls [P = 0.017; odds ratios (OR), 1.71; 95% confidence intervals (95% CI), 1.10-2.66]. There is also a higher frequency of A133S in patients with higher familial breast cancer risk (P = 0.029; OR, 1.76; 95% CI, 1.06-2.92) and patients carrying BRCA1/2 mutations (P = 0.037, OR, 1.82; 95% CI, 1.04-3.18). Importantly, we found that the co-occurrence of a BRCA1 or BRCA2 mutation and A133S in RASSF1A was associated with earlier onset of breast cancer compared with those individuals with either a BRCA1/2 mutation or the A133S polymorphism alone (36.0 versus 42.0 years old, P = 0.002). Our data suggest that the presence of the RASSF1A A133S polymorphism is associated with breast cancer pathogenesis in general and modifies breast cancer age of onset in BRCA1/2 mutations carriers. Our results warrant a large-scale study to examine the effect of the A133S polymorphism in the development of breast and other types of cancers.

Steroid receptor coactivator-3 expression in lung cancer and its role in the regulation of cancer cell survival and proliferation.

Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non-small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA-mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer.

Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts

Purpose: To investigate the frequency of xenotropic murine leukemia virus (MLV) presence in human cell lines established from mouse xenografts and to search for the evidence of horizontal viral spread to other cell lines. Methodology: We examined xenograft tumor cell lines from 7 independent laboratories and 128 non-xenografted tumor cell lines. Cell line DNA was examined for mouse DNA contamination, and by three Taqman qPCR assays targeting the gag, env or pol regions of MLV. Sequencing was used for viral strain identification. Supernatant fluids were tested for reverse transcriptase (RT) activity. Results: Six of 23 (26%) mouse DNA free xenograft cultures were strongly positive for MLV and their sequences had greater than 99% homology to known MLV strains. Four of five available supernatant fluids from these viral positive cultures were strongly positive for RT activity. Three of these supernatant fluids were studied to confirm the infectivity of the released virions for other human culture cells. Of the 78 non-xenograft derived cell lines maintained in the xenograft culture-containing facilities, 13 (17%) were positive for MLV, including XMRV, a virus strain first identified in human tissues. By contrast, all 50 cultures maintained in a xenograft culture-free facility were negative for viral sequences. Conclusions: Human cultures derived after mouse xenografting frequently contain and release highly infectious xenotropic MLV viruses. Laboratories working with xenograft-derived human cultures should be aware of the risk of contamination with potentially biohazardous human-tropic mouse viruses and their horizontal spread to other cultures.