Inhibition of IGF1R enhances 2-deoxyglucose in the treatment of non-small cell lung cancer.

OBJECTIVE: We previously postulated that 2-deoxyglucose (2-DG) activates multiple pro-survival pathways through IGF1R to negate its inhibitory effect on glycolysis. Here, we evaluated whether IGF1R inhibitor synergizes with 2-DG to impede the growth of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: The activation of IGF1R signaling was assessed by the phosphorylation of IGF1R and its downstream target AKT using immunoblot. Drug dose response and combination index analyses were carried out according to the method of Chou and Talalay. Flow cytometry was used to evaluate cell cycle progression. Apoptosis was monitored by caspase-3/PARP cleavages or Annexin V staining. A subcutaneous xenograft model was used to assess this combination in vivo. RESULTS: 2-DG induces the phosphorylation of IGF1R in its kinase domain, which can be abolished by the IGF1R inhibitor BMS-754807. Furthermore, the combination of 2-DG and BMS-754807 synergistically inhibited the survival of several non-small cell lung cancer (NSCLC) cell lines both in vitro and in vivo. The mechanistic basis of this synergy was cell line-dependent, and LKB1-inactivated EKVX cells underwent apoptosis following treatment with a subtoxic dose of 2-DG and BMS-754807. For these cells, the restoration of LKB1 kinase activity suppressed apoptosis induced by this combination but enhanced G1 arrest. In H460 cells, the addition of 2-DG did not enhance the low level of apoptosis induced by BMS-754807. However, treatment with 0.75 µM of BMS-754807 resulted in the accumulation of H460 cells with 8n-DNA content without affecting cell density increases. Hence, H460 cells may escape BMS-754807-induced G2/M cell cycle arrest through polyploidy. The inclusion of 2-DG blocked formation of the 8n-DNA cell population and restored G2/M phase cell cycle arrest. CONCLUSION: The combination of 2-DG and IGF1R inhibitor BMS-754807 may be used to suppress the proliferation of NSCLC tumors through different mechanisms.

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status.

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 (krasG12D/wt/p53-/-/lkb1wt/wt) and t2 (krasG12D/wt/p53-/-/lkb1-/-) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer.

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

Re-expression of LKB1 in LKB1-mutant EKVX cells leads to resistance to paclitaxel through the up-regulation of MDR1 expression.

OBJECTIVES: The tumor suppressor LKB1 has recently been shown to be involved in the regulation of microtubule dynamics, thus cancer cells with inactivated LKB1 may have developed a means to overcome dysregulated microtubule functions, making them intrinsically resistant to microtubule targeting agents. Here, we generated isogenic LKB1-wild type and mutant non-small cell lung cancer (NSCLC) cell lines to evaluate the role of LKB1 in paclitaxel resistance. MATERIALS AND METHODS: SRB, flow cytometry and immunoblotting were used to assess cell proliferation and apoptosis in NSCLC cell lines after paclitaxel treatment. Expression of LKB1 was restored in LKB1-null cells by retrovirus infection and was reduced in LKB1-wild type cells by shRNA knock down. RESULTS AND CONCLUSION: The restoration of LKB1 in LKB1-null cells failed to promote paclitaxel-induced apoptosis in both p53-wild type and p53-mutant backgrounds, indicating that LKB1 was not required for paclitaxel-induced apoptosis. Interestingly, the re-establishment of LKB1 expression led to the up-regulation of class III beta-tubulin and MDR1 in EKVX cells. The up-regulation of MDR1 protein and transcripts in EKVX cells was specifically associated with the expression of wild-type LKB1 and mainly responsible for the increased cellular resistance to paclitaxel. However, the presence of LKB1 protein was not required to maintain this increased MDR1 expression even though there was no genetic amplification or promoter de-methylation of the ABCB1 locus in EKVX-LKB1-WT cells. These data suggest that LKB1 does not promote paclitaxel-induced apoptosis in most NSCLC cell lines. In contrast, in some NSCLC, the presence of LKB1 may facilitate increases in either MDR1 or class III beta-tubulin expression which can lead to paclitaxel resistance.

Primary gastric malignant melanoma: challenge in preoperative diagnosis.

Primary gastric melanoma is an extremely rare clinical entity. The clinical manifestation is not specific and usually similar with other common malignancies at this site, such as gastric cancer and lymphoma. And there are no specific radiological features either. Preoperative diagnosis via biopsy is usually difficult, since melanoma pigment could be absent in the biopsy tissue. Here, we report a case of a 50-year-old woman with a mass in the stomach found by gastroscopy. Biopsy was taken twice preoperatively under gastroscope and it was diagnosed as gastric carcinoma and neuroendocrine tumor respectively. Radical surgery was performed with gastrectomy and D2 lymph node dissection. Postoperative pathological examination finally made a definite diagnosis of gastric melanoma by immunohistochemistry. We summarize the reasons for preoperative misdiagnosis and discuss the difficulty in diagnosing gastric melanoma according to literature.

Evaluation of para-aortic nodal dissection for locoregionally advanced gastric cancer with 1-3 involved para-aortic nodes.

BACKGROUND: Prophylactic para-aortic nodal dissection (PAND) has no proven benefits for potentially curable advanced gastric cancer. However, the value of therapeutic PAND for involved para-aortic nodes (PANs) in patients with locally advanced gastric cancers has not been determined yet. METHODS: Between 1998 and 2010, 157 gastric cancer patients with 1-3 involved PANs underwent extended D2 (D2+) lymphadenectomy plus PAND (PAND group, n = 69) or extended D2 lymphadenectomy alone (non-PAND group, n = 88). The clinicopathologic features and prognostic data were compared between the two groups. A propensity score-adjusted analysis was used for a balanced comparison. RESULTS: The rate of PAN metastasis was 40.6% (28/69) in the PAND group. The 5-year survival rate was significantly higher in the PAND group than in the non-PAND group (43.7% vs. 31.8%, P = 0.044). Compared to the non-PAND group, the death hazard ratios in the PAND group were 0.45 (95% CI 0.274-0.739; P = 0.002) and 0.536 (95% CI 0.328-0.861; P = 0.0097) by multivariate analysis without and with propensity score adjustment respectively. Recurrence rate at 5 years was 39.1% in the PAND group and 43.2% in the non-PAND group (P = 0.628). CONCLUSION: Extended D2 lymphadenectomy plus PAND is associated with superior outcomes for advanced gastric cancer patients with 1-3 involved PANs.

High CpG island methylator phenotype is associated with lymph node metastasis and prognosis in gastric cancer.

Several studies have found that the promoter CpG island is frequently methylated in gastric cancer. The CpG island methylator phenotype (CIMP) defines concordant methylation of multiple promoter CpG island loci in a subset of gastric cancer. However, the relationship between CIMP and lymph node metastasis in gastric cancer is unknown. Our study aimed to characterize the role of CIMP in lymph node metastasis. Clinical specimens from 120 patients were analyzed and PCR was used to detect the methylation status of five genes (ALX4, TMEFF2, CHCHD10, IGFBP3, and NPR1). We measured the level of mRNA for the five genes by real-time RT-PCR. Microsatellite instability and Helicobacter pylori infection status were assayed by capillary electrophoresis and real-time PCR, respectively. DNA methylation in the five genes was correlated with low expression of the respective mRNA. With CIMP as the dependent variable, CIMP-high gastric cancer tended to show more distant lymph node metastasis, higher pathologic tumor classification, more pathologic metastasis, and higher pathologic TNM status. Microsatellite instability and H. pylori status were not significant predictors of prognosis. CIMP-high gastric cancer showed significantly worse survival compared with that of CIMP-low/CIMP-negative gastric cancer (P < 0.001). Our results show that there is an association between CIMP status and lymph node metastasis in gastric cancer and CIMP-high was an independent prognostic factor.