Elizabeth T. Chang

Polyamines and Gut Mucosal Homeostasis

The epithelium of gastrointestinal (GI) mucosa has the most rapid turnover rate of any tissue in the body and its integrity is preserved through the dynamic balance between cell migration, proliferation, growth arrest and apoptosis. To maintain tissue homeostasis of the GI mucosa, the rates of epithelial cell division and apoptosis must be highly regulated by various extracellular and intracellular factors including cellular polyamines. Natural polyamines spermidine, spermine and their precursor putrescine, are organic cations in eukaryotic cells and are implicated in the control of multiple signaling pathways and distinct cellular functions. Normal intestinal epithelial growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines also regulate intestinal epithelial cell (IEC) apoptosis. Although the specific molecular processes controlled by polyamines remains to be fully defined, increasing evidence indicates that polyamines regulate intestinal epithelial integrity by modulating the expression of various growth-related genes. In this review, we will extrapolate the current state of scientific knowledge regarding the roles of polyamines in gut mucosal homeostasis and highlight progress in cellular and molecular mechanisms of polyamines and their potential clinical applications.

Heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by increasing protein translation of selected transcripts in cancer cells

The heterogenous ribonucleoprotein A18 (hnRNP A18) promotes tumor growth by coordinating the translation of selected transcripts associated with proliferation and survival. hnRNP A18 binds to and stabilizes the transcripts of pro-survival genes harboring its RNA signature motif in their 3′UTRs. hnRNP A18 binds to ATR, RPA, TRX, HIF-1α and several protein translation factor mRNAs on polysomes and increases de novo protein translation under cellular stress. Most importantly, down regulation of hnRNP A18 decreases proliferation, invasion and migration in addition to significantly reducing tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, tissue microarrays performed on human melanoma, prostate, breast and colon cancer indicate that hnRNP A18 is over expressed in 40 to 60% of these malignant tissue as compared to normal adjacent tissue. Immunohistochemistry data indicate that hnRNP A18 is over expressed in the stroma and hypoxic areas of human tumors. These data thus indicate that hnRNP A18 can promote tumor growth in in vivo models by coordinating the translation of pro-survival transcripts to support the demands of proliferating cells and increase survival under cellular stress. hnRNP A18 therefore represents a new target to selectively inhibit protein translation in tumor cells.

Loss of p53, rather than beta-catenin overexpression, induces survivin-mediated resistance to apoptosis in an esophageal cancer cell line

OBJECTIVE Survivin, an important inhibitor of apoptosis, is overexpressed in esophageal cancer and negatively affects survival. The complex regulation of survivin transcription involves enhancement by beta-catenin and repression by p53. The purpose of this study is to test whether inhibition of beta-catenin or overexpression of p53 can decrease survivin expression and render esophageal cancer cells more susceptible to apoptosis. METHODS Studies were performed in normal human esophageal epithelial cells and the human esophageal cancer cell line TE7. Levels of beta-catenin, survivin, and p53 were measured by Western blot. Apoptosis was induced after treatment with camptothecin and measured by release of caspase 3 and morphologic criteria. The roles of survivin and beta-catenin in preventing apoptosis were tested by their silencing with specific small interfering RNA molecules. The effect of p53 overexpression on survivin promoter activity was measured using a survivin promoter-luciferase reporter construct and by real-time polymerase chain reaction measurement of survivin mRNA levels. RESULTS Both beta-catenin and survivin are overexpressed in TE7 cells, whereas p53 expression is negligible. TE7 cells demonstrate resistance to camptothecin-induced apoptosis (P < .01). This effect is significantly reduced by inhibition of survivin, but not of beta-catenin (P < .01). Overexpression of p53 in TE7 cells reduces survivin transcription and mRNA levels (P < .01), without reducing survivin protein levels. CONCLUSION Survivin plays a critical role in TE7 cell resistance to camptothecin-induced apoptosis. This effect is not dependent on beta-catenin expression. Overexpression of p53 decreases survivin transcription but does not decrease levels of survivin protein, suggesting posttranscriptional control of survivin expression.

Contribution of Dual Oxidase 2 (DUOX2) to Hyper-Radiosensitivity in Human Gastric Cancer Cells

Whole-abdominal radiotherapy (WART) is a primary method for managing gastrointestinal cancers that have disseminated into intra-abdominal tissues. While effective, this approach is limited because of the increased toxicity to normal tissue associated with combined WART and full-dose chemotherapy regimens. Recent studies have demonstrated a survival advantage in a novel treatment paradigm that allows for the safe use of full-dose systemic chemotherapy in combination with low-dose fractionated radiotherapy (LDFRT). Traditionally, radiation doses greater than 120 cGy have been used in radiotherapy because lower doses were thought to be ineffective for tumor therapy. However, we now know that LDFRT can produce hyper-radiosensitivity (HRS), a phenomenon where cells undergo apoptosis at radiation doses as low as 15 cGy, in a number of proliferating cells. The objectives of our current study were to determine whether LDFRT can induce HRS in gastrointestinal cancer cells and to identify biomarkers of chemopotentiation by LDFRT. Our data indicate that three consecutive daily fractions of 15 cGy produced HRS in gastric cancer cells and potentiated a modified regimen of docetaxel, cisplatin and 5′-fluorouracil (mDCF). Colony survival assays indicated that 15 cGy was sufficient to kill 90% of the cells when LDFRT was combined with mDCF whereas a dose almost 10 times higher (135 cGy) was needed to achieve the same rate when using conventional radiotherapy alone. RT2 PCR Profiler™ array analysis indicated that this combined regimen upregulated dual oxidase 2 (DUOX2), an enzyme functioning in the production of hydrogen peroxide, without upregulating genes involved in DNA repair. Moreover, downregulation of DUOX2 increased radioresistance at every radiation dose tested. In addition, our data indicate that reactive oxygen species (ROS) increase up to 3.5-fold in cells exposed to LDFRT and mDCF. Furthermore, inhibition of NADPH oxidase abrogated the killing efficiency of this combined regimen. Taken together these data suggest that chemopotentiation by LDFRT in gastric cancer cells may be due, at least in part, to increased ROS production (DUOX2) without upregulation of the DNA repair machinery. These data thus provide a rationale for further explorations of potential clinical applications of LDFRT, such as in WART, as a chemopotentiator for advanced and metastatic gastric cancers.

Abstract 5852: DUOX2, a key player for chemopotentiation by low-dose fractionated radiation therapy in gastric cancer cells

One of the most conventional therapy for solid tumors is radiotherapy. Still, this modality presents a challenge when it comes to managing highly disseminated gastrointestinal cancers due to increased toxicity to surrounding tissues. Recent laboratory and clinical data indicate that Low Dose Fractionated Radiation Therapy (LDFRT) can potentiate systemic chemotherapy and presents the possibility to revisit the concept of Whole Abdominal Radiotherapy (WART) for disseminated intra-abdominal gastric cancers. Earlier work considered LDFRT ineffective for tumor removal but we now know that LDFRT induces hyper-radiosensitivity (HRS) in a number of proliferating cells. We have recently shown that dual oxidase (DUOX2) is a major contributor to induce HRS at radiation doses as low as 0.15 Gy and sensitize human gastric cancer cells to chemotherapy. The aim of our study is to determine the utility of DUOX2 as a potential biomarker for the clinical application of chemopotentiation by LDFRT. First, we performed immunohistochemistry (IHC) on 48 human gastric samples with progressive grades. Our data indicate that only about 50% of human gastric cancers are positive for DUOX2. The reason for DUOX2 variability of expression is not clear but may be linked to inflammation since six of the seven (86%) gastritis samples we examined expressed strong levels of DUOX2 in the surface of epithelial cells. Our data also indicate that expression of DUOX2 significantly increases the levels of macrophages infiltration in tissue expressing DUOX2 as well as in the stroma surrounded by cells expressing DUOX2. This suggests that expression of DUOX2 could impact on the dynamic of the tumor microenvironment. Expression of DUOX2 in response to LDFRT is conserved since we also observed this in mice primary gastric cancer cells as well as cancer stem cells. Furthermore, as a first step to develop DUOX2 as an accessible biomarker, we studied DUOX2 activity by measuring the accumulation of oxidative serum proteins in gastric cancer cells media. Our data indicate that down regulation of DUOX2 significantly reduces the levels of serum protein oxidation. Taken together these data suggest that DUOX2 could potentially be used as a biomarker to stratify patients and follow the efficiency of clinical application of chemopotentiation by LDFRT. Citation Format: Palak R. Parekh, Elizabeth Chang, Navesh K. Sharma, France Carrier. DUOX2, a key player for chemopotentiation by low-dose fractionated radiation therapy in gastric cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5852. doi:10.1158/1538-7445.AM2017-5852

Abstract 2796: Regulation of HIF-1α by hnRNP A18 contributes to tumor promotion under hypoxic conditions

The RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) is a new protein translation regulator found to be elevated in many cancers. Previous work from our lab has shown that hnRNP A18 is able to bind and regulate the translation of a group of mRNAs under stress conditions, including hypoxia. Hypoxia has largely been recognized as an innate characteristic of solid tumors and plays numerous cellular and physiologic roles such as cell proliferation, invasion, migration, metastasis, and angiogenesis. Given its extensive function in solid tumors, combined with computational results showing a possible hnRNP A18 signature motif in the 3’UTR of HIF-1α, we sought to determine if hnRNP A18 binds HIF-1α mRNA and how this affects tumor promotion and growth. Results: Our data indicates that hnRNP A18 protein is able to bind HIF-1α mRNA. Under hypoxic conditions, down-regulation of hnRNP A18 significantly decreases HIF-1α mRNA stability, leading to reduced amounts of HIF-1α protein levels in melanoma cells. Treatment with CoCl2 increases hnRNP A18 protein levels in melanoma cells, but not in normal melanocytes. Additionally, colony survival assay demonstrates that down-regulation of hnRNP A18 decreases cells’ survival rates under a hypoxic environment. The functional significance of this effect was observed in a mouse xenograft model where down-regulation of hnRNP A18 significantly reduced melanoma and breast tumor growth. Immunohistochemistry staining of xenograft specimens showed overlapping localization of hnRNP A18 to the tumor hypoxic regions. Lastly, proteome profiling of the xenograft tumors showed a significant decrease of key angiogenic-related factors (e.g., angiogenin, endoglin, VEGF) when hnRNP A18 is reduced. Conclusion: Heterogenous ribonucleoprotein A18 is able to regulate HIF-1α mRNA and modulate its protein levels under hypoxic conditions. The oxygen-deprived regions generated in the central core of a solid tumor leads to up-regulation of hnRNP A18 and consequently HIF-1α, resulting in tumor growth and promotion. New therapeutics targeting hnRNP A18 would therefore be a logical next-step in developing a novel mechanism-based therapy for cancer treatment. Citation Format: Elizabeth T. Chang, Palak Parekh, Qingyuan Yang, France Carrier. Regulation of HIF-1α by hnRNP A18 contributes to tumor promotion under hypoxic conditions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2796.

Abstract 5081: hnRNP A18: an emerging novel target for cancer therapy

Heterogenous ribonucleoprotein A18 (hnRNP A18) increases de novo protein translation during cellular stress. Although hnRNP A18 has recently been associated with several cancers, the exact role of it during the multifactorial process of cancer growth and progression is still unknown. Accordingly, we examined the dependability of various malignant cancer cell lines on hnRNP A18. Down-regulation of hnRNP A18 impaired the proliferative, invasive and migratory properties of melanoma and triple negative breast cancer cell lines in vitro, while overexpression of hnRNP 18 caused an increase in invasiveness. Consistent with these observations, we detected remarkable reduction of tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, down-regulation of hnRNP A18 increased the sensitivity of melanoma cells and breast cancer cells to paclitaxel and 5-fluoro uracil, respectively. In conclusion, hnRNP A18 plays a key role in tumor growth and progression. It may serve as a novel target for the treatment of cancer. Citation Format: Palak R. Parekh, Elizabeth Chang, Qingyuan Yang, France Carrier. hnRNP A18: an emerging novel target for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5081.