Michael J. Pennison

Germline Allele-specific Expression of TGFBR1 Confers an Increased Risk of Colorectal Cancer

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor–β (TGF-β) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-β signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.

Cancer cell proliferation is inhibited by specific modulation frequencies.

Background:There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields.Methods:To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12u2009MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100u2009Hz–21u2009kHz range as cancer-specific frequencies.Results:The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle.Conclusion:These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology.

Targeting transforming growth factor-β signaling

Purpose of review Most cancers are characterized by excessive transforming growth factor-β production by tumors, which can promote tumor growth and mediate epithelial-to-mesenchymal transition. Transforming growth factor-β also has the ability to overproduce extracellular matrix components in response to injury and other stimuli. There are many strategies undergoing current evaluation for inhibiting the deleterious biological effects of transforming growth factor-β by disrupting its signaling at various levels. The current review focuses on the recent advances made in this area, and the potential of these strategies in the clinical treatment of cancer and fibrosis. Recent findings Four main strategies used most recently for disrupting transforming growth factor-β signaling are brought into focus in this review: inhibition or sequestration of the transforming growth factor-β protein ligands, inhibition of transforming growth factor-β receptor kinase activity, inhibition of SMAD signaling downstream of transforming growth factor-β kinase activity and restoration of antitumor immunity upon transforming growth factor-β inhibition. Various techniques currently used to employ these four strategies are discuussed. Summary Several lines of evidence suggest that altered transforming growth factor-β signaling contributes to tumor progression and metastasis as well as development of fibrosis. Accumulating data from preclinical and clinical studies indicate that antagonizing aberrant transforming growth factor-β signaling is a promising novel therapeutic approach in cancer and fibrotic disorders.

Tgfbr1 haploinsufficiency is a potent modifier of colorectal cancer development

Transforming growth factor-beta (TGF-beta) signaling is frequently altered in colorectal cancer. Using a novel model of mice heterozygous for a targeted null mutation of Tgfbr1 crossed with Apc(Min/+) mice, we show that Apc(Min/+);Tgfbr1(+/-) mice develop twice as many intestinal tumors as Apc(Min/+);Tgfbr1(+/+) mice, as well as adenocarcinoma of the colon, without loss of heterozygosity at the Tgfbr1 locus. Decreased Smad2 and Smad3 phosphorylation and increased cellular proliferation are observed in the colonic epithelium crypts of Apc(Min/+); Tgfbr1(+/-) mice. Smad-mediated TGF-beta signaling is preserved in both Apc(Min/+);Tgfbr1(+/+) and Apc(Min/+);Tgfbr1(+/-) intestinal tumors, but cyclin D1 expression and cellular proliferation are significantly higher in Apc(Min/+);Tgfbr1(+/-) tumors. These results show that constitutively reduced Tgfbr1-mediated TGF-beta signaling significantly enhances colorectal cancer development and results in increased tumor cell proliferation. These findings provide a plausible molecular mechanism for colorectal cancer development in individuals with constitutively altered TGFBR1 expression, a recently identified common form of human colorectal cancer.

Targeted treatment of cancer with radiofrequency electromagnetic fields amplitude-modulated at tumor-specific frequencies

In the past century, there have been many attempts to treat cancer with low levels of electric and magnetic fields. We have developed noninvasive biofeedback examination devices and techniques and discovered that patients with the same tumor type exhibit biofeedback responses to the same, precise frequencies. Intrabuccal administration of 27.12 MHz radiofrequency (RF) electromagnetic fields (EMF), which are amplitude-modulated at tumor-specific frequencies, results in long-term objective responses in patients with cancer and is not associated with any significant adverse effects. Intrabuccal administration allows for therapeutic delivery of very low and safe levels of EMF throughout the body as exemplified by responses observed in the femur, liver, adrenal glands, and lungs. In vitro studies have demonstrated that tumor-specific frequencies identified in patients with various forms of cancer are capable of blocking the growth of tumor cells in a tissue- and tumor-specific fashion. Current experimental evidence suggests that tumor-specific modulation frequencies regulate the expression of genes involved in migration and invasion and disrupt the mitotic spindle. This novel targeted treatment approach is emerging as an appealing therapeutic option for patients with advanced cancer given its excellent tolerability. Dissection of the molecular mechanisms accounting for the anti-cancer effects of tumor-specific modulation frequencies is likely to lead to the discovery of novel pathways in cancer.

Prevention and treatment of cancer with aspirin: where do we stand?

Aspirin is arguably the synthesized drug that has been used most commonly in human history. Aspirin was originally developed and marketed for the treatment of inflammatory disorders at the end of the 19th century, but its mechanism of action remained unknown until the second half of the 20th century. Since the latter part of the 20th century aspirin also has been used for the primary and secondary prevention of cardiovascular diseases given its anti-thrombotic properties. An association between intake of aspirin and decreased cancer risk was identified in the past decades. Whether aspirin can be used as an anticancer agent in patients with a diagnosis of cancer was unknown until recently. Recent studies suggest that aspirin might provide therapeutic benefit in the adjuvant treatment of certain forms of cancer. This review provides a critical update on this topic, which has potential implications for oncologists and their patients.

Constitutively decreased TGFBR1 allelic expression is a common finding in colorectal cancer and is associated with three TGFBR1 SNPs

PurposeConstitutively decreased TGFBR1 allelic expression is emerging as a potent modifier of colorectal cancer risk in mice and humans. This phenotype was first observed in mice, then in lymphoblastoid cell lines from patients with microsatellite stable colorectal tumors.Patients and MethodsWe assessed the frequency of constitutively decreased TGFBR1 allelic expression and association with SNPs covering the TGFBR1 locus using RNA and DNA extracted from the peripheral blood lymphocytes of 118 consecutive patients with biopsy-proven adenocarcinoma of the colon or the rectum.ResultsWe found that 11(9.3%) of 118 patients exhibited decreased TGFBR1 allelic expression (TGFBR1 ASE). TGFBR1 ASE was strongly associated with three SNPs in linkage disequilibrium with each other: rs7034462 (p = 7.2 × 10-4), TGFBR1* 6A (p = 1.6 × 10-4) and rs11568785 (p = 1.4 × 10-4).ConclusionThese results confirm the high prevalence of constitutively decreased TGFBR1 allelic expression among patients with colorectal cancer. The association of this phenotype with TGFBR1*6A, rs7034462 and rs1156875 suggests an association between TGFBR1 SNPs and colorectal cancer, which warrants additional studies.

Characterization of the colorectal cancer-associated enhancer MYC-335 at 8q24: The role of rs67491583

Recent genome-wide association studies have identified multiple regions at 8q24 that confer susceptibility to many cancers. In our previous work, we showed that the colorectal cancer (CRC) risk variant rs6983267 at 8q24 resides within a TCF4 binding site at the MYC-335 enhancer, with the risk allele G having a stronger binding capacity and Wnt responsiveness. Here, we searched for other potential functional variants within MYC-335. Genetic variation within MYC-335 was determined in samples from individuals of European, African, and Asian descent, with emphasis on variants in putative transcription factor binding sites. A 2-bp GA deletion rs67491583 was found to affect a growth factor independent (GFI) binding site and was present only in individuals with African ancestry. Chromatin immunoprecipitation performed in heterozygous cells showed that the GA deletion had an ability to reduce binding of the transcriptional repressors GFI1 and GFI1b. Screening of 1,027 African American colorectal cancer cases and 1,773 healthy controls did not reveal evidence for association (odds ratio: 1.17, 95% confidence interval: 0.97-1.41, P = 0.095). In this study, rs67491583 was identified as another functional variant in the CRC-associated enhancer MYC-335, but further studies are needed to establish the role of rs67491583 in the colorectal cancer predisposition of African Americans.

Lynch Syndrome Testing: A Missed Opportunity in the Era of Precision Medicine

Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer, is an autosomal-dominant inherited cancer susceptibility syndrome that affects approximately 1 in 35 patients diagnosed with colorectal or endometrial cancers, making it the most common inherited cancer syndrome.1 Lifetime risk of colorectal cancer for men and women with Lynch syndrome is 10% to 80%, and women with Lynch syndrome have a 15% to 60% lifetime risk of developing endometrial cancer. Lynch syndrome is caused by inactivating germline mutations of 1 of 4 mismatch repair genes (mutL homolog 1 [MLH1], MutS homolog 1 [MSH2], MutS homolog 6 [MSH6], and PMS 1 homolog 2 [PMS2])2 or the epithelial cell adhesion molecule (EPCAM) gene located upstream of the MSH2 gene.3 These genes are essential for correcting nucleotide base mispairs and small insertions or deletions that may occur because of misincorporations or DNA polymerase errors during DNA replication.4 As tumors develop in individuals with Lynch syndrome, the function of the normal (wild-type) allele inherited from the unaffected parent is lost, resulting in a loss of mismatch repair function. In the absence of mismatch repair, simple repetitive sequences (microsatellites) accumulate throughout the genome, resulting in microsatellite instability.4 Colorectal and endometrial cancers are preventable through the use of agents such as aspirin (chemoprevention),5 cancer surveillance,6 and risk-reducing prophylactic surgery options.7 Identifying Lynch syndrome can prevent cancers and reduce the mortality risk in affected families.8 However, traditional use of clinical criteria such as the Amsterdam II9 and the revised Bethesda criteria,10 as well as genetic modeling to identify patients with Lynch syndrome, have been criticized for being inefficient and having less than optimal sensitivity.11 Under current National Comprehensive Cancer Network (NCCN) clinical testing criteria, genetic testing for Lynch syndrome among patients with colorectal and endometrial cancer is only recommended for individuals younger than 50 years who meet the Amsterdam II criteria or the revised Bethesda guidelines and for individuals with a known family history of Lynch syndrome.12 However, studies evaluating the efficacy of universal molecular testing of colorectal cancers reveal that as many as 28% of individuals with Lynch syndrome are not identified using even the most liberal clinical criteria under the revised Bethesda guidelines.4 Similarly, evidence suggests that universal screening for Lynch syndrome among patients with newly diagnosed endometrial cancer may significantly increase Lynch syndrome identification among patients and their relatives.13 In this issue of JAMA, Usha and colleagues14 describe the case of a 43-year-old woman diagnosed with stage IA endometrial cancer. Following surgical resection, her tumor was screened for Lynch syndrome using a panel of 4 mismatch repair protein markers by immunohistochemistry staining, which revealed the absence of 1 of these 4 markers. Subsequent genetic testing revealed a germline mutation in the MSH6 gene, establishing the diagnosis of Lynch syndrome. The patient was advised to undergo increased cancer surveillance with a colonoscopy every 1 to 2 years, and genetic testing was advised for first-degree relatives. Because the patient was younger than 50 years, she met the NCCN criteria for Lynch syndrome testing.12 However, individuals older than aged 50 years are currently ineligible for Lynch syndrome testing under the NCCN criteria, regardless of family history, even if tumor screening is performed showing suspicion of Lynch syndrome. If this patient had been older than aged 50 years, this diagnosis could have been missed and she would not have received increased cancer surveillance. Patients with Lynch syndrome also have an increased risk of developing malignancies of the stomach, ovaries, hepatobiliary tract, urinary tract, small bowel, central nervous system, pancreas, prostate, and breast15 and can be counseled regarding tumor-specific screening such as urinalysis to screen for tumors of the urinary tract and preventive measures such as salpingo-oophorectomy to prevent ovarian cancer. Because mutations associated with Lynch syndrome are inherited through germline transmission in an autosomaldominant fashion, patients with tumors for which Lynch Syndrome is suspected should be referred for genetic counseling, regardless of their family history. If Lynch syndrome is confirmed, genetic counseling and testing should be offered to all first-degree relatives aged 18 years and older because they have a 50% chance of inheriting the mutated mismatch repair gene. In these relatives, testing for the exact Lynch syndrome gene(s) and mutation(s) responsible can be implemented according to the mutation of the index patient. This targeted approach makes testing less expensive.16 Three interrelated issues should be considered when considering universal Lynch syndrome testing. The first is which testing platforms should be used. Most testing methods are widely available in a large number of pathology pracRelated article page 93 Opinion

Dual role of TGFBR1 as a modifier of colorectal cancer risk.

600 Background: Experimental and clinical evidence suggests that constitutively decreased Transforming Growth Factor Beta type I receptor (TGFBR1) signaling predisposes to colorectal cancer (CRC) development. However, associations between TGFBR1 variants and CRC risk in case-control studies have been inconsistent. Methods: We utilized 1,043 CRC cases and their 1,627 unaffected sibling controls obtained from the Colon Cancer Family Registry (C-CFR). Individuals were genotyped for twelve TGFBR1 haplotype tagging SNPs. SNPs associated with CRC risk were validated in 261 CRC cases and 531 controls of African American ancestry and 990 CRC cases and 3,427 controls of Han Chinese ancestry. Validated SNPs were functionally characterized with respect to TGFBR1 expression and TGF-β signaling. Results: The TGFBR1 rs7034462-TT genotype was associated with CRC risk in C-CFR participants (OR 3.80[1.46-9.85]) and African Americans (OR 8.16[2.07-32.08]) (see Table). The TT genotype was associated with stage III and stage...