pISSN 1598-2998, eISSN 2005-9256 Cancer Res Treat. 2017;49(2):387-398 https://doi.org/10.4143/crt.2016.106 Original Article Open Access Underexpression of HOXA11 Is Associated with Treatment Resistance and Poor Prognosis in Glioblastoma Young-Bem Se, MD1 Purpose Seung Hyun Kim, MD2 Homeobox (HOX) genes are essential developmental regulators that should normally be in the silenced state in an adult brain. The aberrant expression of HOX genes has been asso- Ji Young Kim, MS1 ciated with the prognosis of many cancer types, including glioblastoma (GBM). This study PhD1 Ja Eun Kim, examined the identity and role of HOX genes affecting GBM prognosis and treatment 1 Yun-Sik Dho, MD resistance. Jin Wook Kim, MD, PhD1 Yong Hwy Kim, MD1 Materials and Methods The full series of HOX genes of five pairs of initial and recurrent human GBM samples were Hyun Goo Woo, MD, PhD3 screened by microarray analysis to determine the most plausible candidate responsible for MD, PhD4 Se-Hyuk Kim, GBM prognosis. Another 20 newly diagnosed GBM samples were used for prognostic vali- 5 Shin-Hyuk Kang, MD, PhD dation. In vitro experiments were performed to confirm the role of HOX in treatment resist- 6 Hak Jae Kim, MD, PhD ance. Mediators involved in HOX gene regulation were searched using differentially Tae Min Kim, MD, PhD7 expressed gene analysis, gene set enrichment tests, and network analysis. Soon-Tae Lee, MD, PhD8 Results MD, PhD9 Seung Hong Choi, The underexpression of HOXA11 was identified as a consistent signature for a poor prog- 10 Sung-Hye Park, MD, PhD nosis among the HOX genes. The overall survival of the GBM patients indicated a signifi- 6 Il Han Kim, MD, PhD cantly favorable prognosis in patients with high HOXA11 expression (31±15.3 months) Dong Gyu Kim, MD, PhD1,2 compared to the prognoses in those with low HOXA11 expression (18±7.3 months, p=0.03). Chul-Kee Park, MD, PhD1,2 When HOXA11 was suppressed in the GBM cell lines, the anticancer effect of radiotherapy and/or temozolomide declined. In addition, five candidate mediators (TGFBR2, CRIM1, *A list author’s aliations appears at the end TXNIP, DPYSL2, and CRMP1) that may confer an oncologic effect after HOXA11 suppression of the paper. were identified. Conclusion + C +o r+re s+p o +n d +e n +c e :+ D +o n +g G +y u+ K +im +, M + D +, P +h D + + + + + + + + + + + + + + + + + + + + + + + The treatment resistance induced by the underexpression of HOXA11 can contribute to a + D +e p+a r +tm +en +t o +f N +e u +ro s+u r+g e +ry ,+ S e+o u +l N +a t+io n+a l+ + poor prognosis in GBM. Further investigation will be needed to confirm the value of HOXA11 University Hospital, Seoul National University + + + + + + + + + + + + + + + + + + + + as a potential target for overcoming the treatment resistance by developing chemo- or radio- College of Medicine, 101 Daehak-ro, + + + + + + + + + + + + + + + + + + + + sensitizers. + J o+n g+n o+- g +u , +S e +ou +l 0 3+0 8 +0 , +K o +re a+ + + + + + + + + T +e l :+ 8 2 +-2 -+2 0 +72 -+2 8 +74 + + + + + + + + + + + + + F +a x +: 8 2+- 2 +-7 4+1 - 8+9 5+4 + + + + + + + + + + + + + E +-m +a i l+: g +k n +if e +@ s +n u +.a c +.k r+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Key words + C +o -+co r+r e +sp o+n d+e n+c e +: C +h u +l- K +e e+ P a+r k +, M +D +, P +h D + + Homeobox genes, HOXA11, Glioblastoma, Treatment resistance + D +e p +a r +tm +en +t o +f N +e u +ro s+u r+g e +ry ,+ S e+o u +l N +a t+io n+a l+ + + U +n i+v e +rs i t+y H+ o +sp +it a +l, S +e o +u l +N a +ti o +n a +l U +n i +ve r+s i t+y + + + + + + + + + + + + + + + + + + + + + + C +o l+le g +e o+f M + e +d i c+in e+, 1 +0 1 + D a+e h+a k +-r o+, + + + + + + J o+n g+n o+- g +u , +S e +ou l+ 0 3+0 8 +0 , +K o +re a+ + + + + + + + + T +e l :+ 8 2 +-2 -+2 0 +72 -+0 3 +47 + + + + + + + + + + + + + F +a x +: 8 2+- 2 +-7 4+1 - 8+5 9+4 + + + + + + + + + + + + + E +-m +a i l+: n +sc k+p a+r k +@ s+n u +.a c+.k r+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + R +e c +ei v +e d + M +a r+c h + 9 , +2 0 +16 + + + + + + + + + + + A +c c +ep +te d + J +un +e 3 +0, 2+0 1+6 + + + + + + + + + + + P +u b +li s +h e d+ O +n l+in e+ J+u l y+ 1 9+, 2+0 1 +6 + + + + + + + + + + + + + + + + + + + + + + + + + + + + * +Y o +u n +g - B+e m+ S+e a+n d+ S +eu n+g +H y+u n+ K +im + + + + + + + + + + + + + + + + + + + + + + + + + + c +on +tr i b+u t+e d + e q +u a+ll y + t o + t h +is w+ o +rk +. + + + + + + │ http://www.e-crt.org │ Copyright ⓒ 2017 by the Korean Cancer Association 387 This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Cancer Res Treat. 2017;49(2):387-398 Introduction Materials and Methods Homeobox (HOX) genes are essential developmental reg- 1. Patient samples and cell lines ulators that control a wide range of processes, including apoptosis, differentiation, motility, and angiogenesis [1]. In Fresh frozen tumor tissue samples of five GBM patients, humans, there are four HOX clusters (A, B, C, and D), and in whom of pairs of initial and recurrent samples were avail- 39 HOX genes have been identified [1]. The HOX genes are able for screening, and another 20 newly diagnosed GBM normally active during embryogenesis, but most are not patients for validation were used in this study. All patients expressed or are expressed at very low levels in an adult were managed using a standard GBM treatment protocol of brain [2]. On the other hand, several studies have indicated concurrent radiotherapy and TMZ treatment, followed by aberrant expression of the HOX genes in brain tumors as well adjuvant TMZ as a primary treatment. The tumor tissues in as other cancers from various organs [3-6]. Moreover, there were obtained during surgery, snap-frozen in liquid nitro- is growing clinical evidence of a prognostic effect of HOX gen, and stored at 80°C prior to use. The study was gene expression in several cancers [7-10]. approved by an institutional review committee. Aberrantly expressed HOX genes in cancer cells have mul- The human glioma U251, U373, and LN18 cell lines were ticapacity functions, including metastasis, tumor growth, purchased from the American Type Culture Collection anti-apoptosis, and differentiation suppression [1]. The over- (Manassas, VA), and cultured in Dulbecco's modified Eagle's expression of multiple HOX genes have been observed in medium containing 10% fetal bovine serum and 5% antibi- glioblastoma (GBM) cell lines and primary astrocytoma [4]. otics (streptomycin) in a humidified atmosphere containing In addition, some studies have shown that the HOX genes 5% CO2 and 95% air at 37°C. are important in the treatment resistance of GBM [11-13]. On the other hand, the precise mechanism showing the role of 2. Reverse transcription polymerase chain reaction HOX genes and their functional relevance in glioma cells is unclear. GBMs, similar to other cancers, harbor a cell sub- The cell lines were lysed with TRIzol (Life Technologies, population with a stem cell-like capacity that is associated Carlsbad, CA), and RNA isolation was performed using an with the development of a tumor progeny and treatment RNeasy Mini Kit (#74104, Qiagen, Valencia, CA). The total resistance [9,14,15]. Given the roles of HOX genes in devel- RNA was treated with DNase and then quantified by spec- opment and organogenesis, it has been postulated that a por- trophotometry. In addition, cDNA was synthesized from tion of the relative expression of HOX genes is integral to 1 µg of the total RNA using a reverse transcription kit stem cell activity, specifically self-renewal, tissue specificity, (#205311, Qiagen) according to the manufacturer’s protocol. and quiescence [1]. Among the HOX genes, the HOXA clus- The primers used were designed using an online primer- ter is important in human embryonic stem cell differentiation BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer- [16]. Collectively, these results suggest that HOX genes are blast/). The primer sequences for HOXA11 were 5'-GATTT- plausible candidates as biomarkers for assessing the progno- CTCCAGCCTCCCTTC-3' (forward) and 5'-AGAAATTG- sis of GBM and a credible target for overcoming the treat- GACGAGACTGCG-3' (reverse). Using these primers, a ment resistance in GBM. reverse transcription polymerase chain reaction (RT-PCR) HOX genes were previously reported to be the genes of was performed for 35 cycles. Each cycle was comprised of interest related to GBM recurrence and treatment resistance 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 45 sec- [17]. Moreover, a mechanism through which the HOXA10 onds with each primer set. The RT-PCR products were gene affects temozolomide (TMZ) resistance in GBM cell resolved by 2% agarose gel electrophoresis. lines was reported [12]. HOXA10 induces the transcription of early growth response 1, which results in phosphatase and 3. Western blot tensin homolong (PTEN) and Rad51 paralogs.