Patterns of TPD52 Overexpression in Multiple Human Solid Tumor Types Analyzed by Quantitative PCR

Patterns of TPD52 Overexpression in Multiple Human Solid Tumor Types Analyzed by Quantitative PCR

INTERNATIONAL JOURNAL OF ONCOLOGY 44: 609-615, 2014 Patterns of TPD52 overexpression in multiple human solid tumor types analyzed by quantitative PCR �IERRE TENNSTE�T1*, CHARLOTTE BÖLCH2*, GUN�ULA STROBEL2, SARAH MINNER2, LIA BURKHAR�T2, TOBIAS GROB2, SAWINEE MASSER2, GUI�O SAUTER2, THORSTEN SCHLOMM1,3 ��� RONAL� SIMON2 1M������-C�����, 2I�������� �f �������gy, 3���������� �f U����gy, S������ f�� T������������ �������� C����� R�������, U��v�����y M������ C����� H��b��g-E�������f, H��b��g, G�����y Received Septemb�� 24, 2013; Accepted Octob�� 29, 2013 �OI: 10.3892/�j�.2013.2200 Abstract. Tumor���� protein������� �52��� �TPD��52��� is�� located������� at�� chromo������- for �xample tumors �f the breast, �vary, endometrium, lung, some 8q21, a region that is frequently gained or amplified colon, head and neck, urinary bladder, kidney and prostate in multiple human cancer �ypes. TPD52 has been suggested �1,2�. Whereas gains �ften �ffect larg� portions �f 8q or as � potential target for new anticancer therapies. I� order even the entire q-arm, high-level amplifications are typically to analyz� TPD52 �xpression in the most prevalent human focused on the chromosomal bands 8q21 �3-5� and 8q24 �6,7�, cancer �ypes, w� employed quantitativ� �CR to measure thus highlighting the loci �f putativ� oncogenes, including TPD52 mRNA levels in formalin-fixed tissue samples from MYC at 8q24. S�veral candidate oncogenes hav� been more than 900 cancer tissues �btained from 29 different suggested to reside inside the 8q21 region, including tumor human cancer �ypes. TPD52 was �xpressed at varying levels protein �52 �TPD52� �3,8-13�. TPD52 has been suggested to in all tested normal tissues, including �kin, �ymph node, lung, play a role for vesicle trafficking and Ca2+ dependent �xocy- oral mucosa, breast, endometrium, �vary, vulv�, �yometrium, totic secretion, and has been show� to facilitate �ytokinesis in liver, pancreas, stomach, kidney, prostate, testis, urinary rapidly proliferating cells �14,15�. bladder, thyroid gland, brain, muscle and fat tissue. TPD52 I� line with an oncogenic role, TPD52 �verexpression has was upregulated in 18/29 �62%� tested cancer �ypes. Strongest been described from � multitude �f cancer �ypes, including �xpression was found in non-seminoma �56-fold �verexpres- breast �9�, lung prostate �2�, �varian �8�, pancreatic cancer �16�, sion compared to corresponding normal tissue), seminoma multiple �yeloma �17,18�, Burkitt'� �ymphoma �19�, melanoma �42-fold), ductal �28-fold) and lobular breast cancer �14-fold). �20� and testicular germ cell tumors �21�, and has been linked I� these tumor �ypes, TPD52 upregulation was found in the to poor prognosis in breast, medulloblastoma, lung and prostate vast majority �>80%� �f tested samples. Downregulation was cancer patients �22�. Moreov��, cell line �xperiments and in vivo found in 11 �38%� tumor �ypes, most strong�y in papillary analysis in mice support the implication �f TPD52 in tumori- renal cell cancer �-8-fold), leiomyosarcoma �-6-fold), clear genesis and progression to metastasis �23,24�. According�y, cell renal cell cancer �-5-fold), liposarcoma �-5-fold) and lung TPD52 has been suggested as � promising target for antitumor cancer �-4-fold). These results demonstrate that TPD52 is therapies in breast �25� and prostate cancer �24�, and it seems frequently and strong�y upregulated in many human cancer likely that also other tumor �ypes showing TPD52 �verexpres- �ypes, which may represent candidate tumor �ypes for poten- sion could profit from a TPD52 specific therapy. tial anti-TPD52 therapies. A �ystematic analysis �f TPD52 �xpression in human cancers in order to identify tumor types that might benefit Introduction from potential anti-TPD52 therapies is lacking. I� this study, w� hav� employed quantitativ� real-time �CR in more than Copy numb�� increase involving chromosome 8q belong to the 900 tumor samples to compare the prevalence and �xpression most frequent �berrations in human solid cancers, including levels �f TPD52 across 29 important human cancer �ypes and corresponding normal tissues. Materials and methods Correspondence to: Dr Ronald Simon, Institute �f Pathology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Tissue specimens. Formalin-fixed, paraffin embedded tissues �-20246 Hamburg, Germany were selected from the archiv� �f the Institute �f Pathology, E-mail: �.simon@�k�.uni-hamburg.de University Medical Center Hamburg-Eppendorf �Hamburg, * Germany�. A total �f 999 cancer samples and 40 normal Contributed �qually tissue samples were included into the study. A detailed list �f all samples is g�ven in T�ble I. One pathologist reviewed Key words: TPD52, cancer, real-time �CR, �RNA analysis, normal tissue all hematoxylin and eosin stained sections �f all tissues and selected one block per tumor for RNA isolation. For tumor 610 TENNSTE�T et al: TPD52 U�REGULATION IN �IFFERENT TUMOR TY�ES T�b�� I. T����� ������� f�� TPD52 �x�������� ����y���. T����� �y�� O�g�� � O�g�� � N����� Sk�� 2 Pancreas 1 Ly��� ���� 2 S������ 2 L��g 2 K����y 2 O��� ��v���y 2 Prostate 2 B����� 1 T����� 3 E���������� 2 B������ 2 Ov�� 2 T�y���� g���� 2 V��v� 2 B���� 2 My�������� 2 Sk������ ������ 2 L�v�� 3 A������ ������ 2 C����� M���g���� �������� 11 L�v�� ������ 50 L��y�x ��������� 39 Pancreatic ������ 38 L��g ������, ���g� ���� �24�, ����� �68�, ����� ���� �17� ��� �q������ ���� �25� 134 S������ ������ 50 O��� ��v��y ������ 56 R���� ���� ������ 59 B����� ������, ������ �26� ��� ��b���� �27� 53 Prostate ������ 48 E���������� ��������� 31 T����� ������, �������� �30� ��� ���-�������� �29� 59 Ov����� ������ 33 U�����y b������ ������, ���-��v���v� ��T� 27� and invasive (≥pT2 28) 55 U����� ���v�x ��������� 28 T�y���� g���� ������ 40 V��v� ������ 39 L����y�������� 42 C���� ������ 50 L���������� 36 E�����g��� ������, �q������ ���� �23� ��� �������������� �25� 48 samples areas with high tumor cell content (≥60% tumor amplification of TPD52 and the housekeeping gene TB� the cells) were marked on the slide. A hollow needle was used QuantiTect Primer Assay �Qiagen) was used. The following to tak� �w� tissue �ylinders (diameter from 0.5 mm) from conditions were used for �CR: i) initial denaturation step at each tissue block for nucleic acid isolation. The local ethics 95˚C for 10 min; and ii) 40 cycles at 95˚C for 20 sec and committee approved usag� �f the human tissue samples for 55˚C for 40 sec. Relative quantity of TPD52 �xpression in research purposes. tumor samples was calculated by the 2-∆∆C� method standard- �zed to TPD52 �xpression in corresponding normal tissue. RNA extraction and cDNA synthesis. Punched tissue mate- A fold chang� �f 2 was used to determine the frequency �f rial was deparaffinized with xylene and 80% ethanol. After significant TPD52 regulated cancers. digestion with proteinases K at 56˚C overnight, total RNA was isolated using the RNeasy FF�E kit �Qiagen) in � full- Results automated nucleic acid isolation device �QIAcub�, Qiagen). cDNA was �ynthesized in � 96-well plate format using the Technical issue. A total �f 894 cancer samples from 29 different high‑capacity cDNA reverse transcription kit �Applied tumor �ypes and 40 normal tissue samples from 20 different Biosystems) following the manufacturer'� instructions. Total normal tissue �ypes were included in the analysis �T�ble I�. A RNA �1 µg� was used for reverse transcription �f all samples. total �f 105 �10.5%� tumor samples and 3 �7.5%� normal tissue samples were �xcluded from analysis because either the ref��- Quantitative PCR analysis. Real-time �CR was performed ence gene TBP or the target gene TPD52 showed � C� value using the L�ghtCycler LC480 �Roche) detection �ystem, and �xceeding 35, indicating that too little cDNA was generated the QuantiTect SYBR-Green PCR Kit (Qiagen). For specific for reliable TPD52 �xpression analysis. INTERNATIONAL JOURNAL OF ONCOLOGY 44: 609-615, 2014 611 F�gure 1. TPD52 �xpression ��vels in different normal tissue. Relativ� TPD52 �xpression ��vel (log2� was standardized to TPD52 �xpression in testis. TPD52 expression in normal tissues. TPD52 transcript was Prevalence of TPD52 expression in cancer. I� order to estimate found in all analyzed normal tissues. Averag� ∆C� was 2.4±0.4. the variability �f TPD52 �xpression in the analyzed tumor �ypes, For � direct comparison �f the TPD52 �xpression levels �f the w� determined the fraction �f samples showing at least 2-fold different normal tissues, the �xpression levels were normalized up- or downregulation. This analysis revealed that TPD52 �ver- to the TPD52 �xpression level in testis, which had the lowest �xpression was particular frequent �80% �f samples showing �xpression level �f all analyzed normal tissues. According�y, ≥2-fold overexpression) in germ cell tumors (non-seminoma highest TPD52 expression levels [≥32 (25� -fold �verexpression and seminoma), bladder cancer, esophageal carcinoma and as compared to normal testis] were found in normal oral cavity mamma carcinoma, whereas cancer �ypes �ypically showing mucosa �104-fold �verexpression), pancreas �91-fold), thyroid TPD52 downregulation included renal cell carcinoma �90% gland �79-fold), kidney �76-fold), prostate �69-fold), stomach papillary and 68% clear cell), leiomyosarcoma (69%� and lipo- �56-fold), vulv� �45-fold), liv�� �39-fold), �ymph node �38-fold), sarcoma �52%�. All data are show� in F�g. 3. and breast �32-fold). All data are summarized in F�g. 1. Discussion TPD52 expression in cancer. T� compare TPD52 �xpression across the different tumor �ypes, �verag�

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