SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

Abstract

Background Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear. Methods Utilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC. Results Analyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease. Conclusions Hemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Independent somatic evolution underlies clustered neuroendocrine tumors in the human small intestine

“…The observation that the most recurrent somatic genetic event in SI-NET, hemizygous chr18 loss, can affect both the maternal and the paternal chromosome homologs in the same patient argues against a contribution from a predisposing chr18 germline variant through loss of heterozygosity 12 . While the tumor suppressors DCC and SMAD4 have been nominated as possible drivers of this event through haploinsufficiency, more work is needed to fully understand the role of chr18 loss in SI-NET 30 , 31 . Furthermore, in stark contrast to germline-induced gastrointestinal tumors, SI-NET only affects a limited intestinal segment.…”

Smad4 and p53 synergize in suppressing autochthonous intestinal cancer

“… 5 Smad4 deficiency promotes intestinal tumorigenesis and metastasis in mice. 6 , 7 , 8 , 9 , 10 Smad4 haploinsufficiency reportedly affects mouse intestinal tumorigenesis and progression and Smad4 deletion in combination with genetic alterations in antigen‐presenting cell (APC) results in intestinal cancer in mice. 9 , 10 Smad4 deletion along with an APC alteration results in intestinal cancer in mice.…”

“… 6 , 7 , 8 , 9 , 10 Smad4 haploinsufficiency reportedly affects mouse intestinal tumorigenesis and progression and Smad4 deletion in combination with genetic alterations in antigen‐presenting cell (APC) results in intestinal cancer in mice. 9 , 10 Smad4 deletion along with an APC alteration results in intestinal cancer in mice. 9 Smad4‐mediated BMP signaling inhibits intestinal tumorigenesis, 6 while Smad4‐independent BMP signaling promotes metastasis in colorectal tumors.…”

Genetic Drivers of Ileal Neuroendocrine Tumors

“…No "second hits" of SMAD2 and SMAD4 have been reported in I-NETs and expression of these genes does not decrease at the mRNA level in I-NETs that have lost chromosome 18 [55]. Decreased SMAD4 expression was reported upon comparing metastatic with localized small intestinal NETs [56].…”