Jacquelyn W. Zimmerman

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 27.12 MHz 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 100 Hz–21 kHz 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.

Molecular mechanisms of Polyphyllin I-induced apoptosis and reversal of the epithelial-mesenchymal transition in human osteosarcoma cells.

Osteosarcoma is a most common highly malignant bone tumor in children and adolescents. Polyphyllin I (PPI) is an ethanol extraction from Paris polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz, which belongs to antipyretic-detoxicate family and has been used as a natural medicine in the treatment of infectious disease and cancer in China for centuries. The proteasome activity inhibitory and anti-osteosarcoma effects of PPI have not been known. Here we found PPI exhibited a selective inhibitory effect on proteasomal chymotrypsin (CT)-like activity, both in purified human proteasome and in cultured osteosarcoma cellular proteasome, and caused an accumulation of ubiquitinated proteins. PPI also inhibited viability, proliferation, migration, and invasion of MG-63, Saos-2, and U-2 OS osteosarcoma cells and resulted in S phase arrest and apoptosis. Furthermore, we explored the molecular targets involved. Exposure of osteosarcoma cells to PPI caused an inactivation of the intrinsic nuclear factor κB (NF-κB) and activation of unfolded protein response (UPR)/endoplasmic reticulum (ER) stress signaling cascade in osteosarcoma cells, followed by down-regulation of anti-apoptotic proteins, with up-regulation of pro-apoptotic proteins. We also demonstrated down-regulation of c-Myc, Cyclin B1, Cyclin D1, and CDK1, which are involved in the cell cycle and growth. Finally, we identified down-regulation of Vimentin, Snail, Slug, and up-regulation of E-cadherin, which are integral proteins involved in epithelial-mesenchymal transition (EMT). Taken together, our data provide insights into the mechanism underlying the anticancer activity of PPI in human osteosarcoma cells.

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.

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.

SMAD4 is a potential prognostic marker in human breast carcinomas

SMAD4 is a downstream mediator of transforming growth factor beta. While its tumor suppressor function has been investigated as a prognostic biomarker in several human malignancies, its role as a prognostic marker in breast carcinoma is still undefined. We investigated SMAD4 expression in breast carcinoma samples of different histologic grades to evaluate the association between SMAD4 and outcome in breast cancer. We also investigated the role of SMAD4 expression status in MDA-MB-468 breast cancer cells in responding to TGF-β stimulation. SMAD4 expression was assessed in 53 breast ductal carcinoma samples and in the surrounding normal tissue from 50 of the samples using immunohistochemistry, Western blot, and real-time PCR. TGF-β-SMAD and non-SMAD signaling was assessed by Western blot in MDA-MB-468 cells with and without SMAD4 restoration. SMAD4 expression was reduced in ductal breast carcinoma as compared to surrounding uninvolved ductal breast epithelia (p < 0.05). SMAD4 expression levels decreased from Grade 1 to Grade 3 ductal breast carcinoma as assessed by immunohistochemistry (p < 0.05). Results were recapitulated by tissue array. In addition, immunohistochemistry results were further confirmed at the protein and mRNA level. We then found that non-SMAD MEK/MAPK signaling was significantly different between SMAD4 expressing MDA-MB-468 cells and SMAD4-null MDA-MB-468 cells. This is the first study indicating that SMAD4 plays a key role in shifting MAPK signaling. Further, we have demonstrated that SMAD4 has a potential role in the development of breast carcinoma and SMAD4 was a potential prognostic marker of breast carcinoma. Our findings further support the role of SMAD4 in breast carcinoma development. In addition, we observed an inverse relationship between SMAD4 levels and breast carcinoma histological grade. Our finding indicated that SMAD4 expression level in breast cancer cells played a role in responding non-SMAD signaling but not the canonic SMAD signaling. Further mechanistic studies are necessary to establish the role of SMAD4 in breast carcinoma prognosis and potential specific targeting.

Abstract 1406: Tgfbr1 haploinsufficiency is a potent modifier of breast cancer risk.

Background: We previously identified a hypomorphic TGF-β type 1 receptor variant (TGFBR1*6A) that is associated with cancer risk and has impaired TGF-β signaling capability. Two recent large meta-analyses of case control studies have found a significant association between TGFBR1*6A and risk of several types of cancer, including breast cancer (ORs 1.16,1.01-1.34; 1.15,1.01-1.31). To investigate the effects of constitutively decreased TGFBR1 signaling on cancer development, we developed a novel mouse model of Tgfbr1 haploinsufficiency to mimic the decreased TGFBR1 signaling observed in individuals with higher risk of cancer. Using this model, we demonstrated that Apcmin;Tgfbr1+/- mice develop more than twice as many intestinal tumors as Apcmin controls. The aim of the current study was to assess the effects of Tgfbr1 haploinsufficiency on breast carcinogenesis by crossing Tgfbr1+/- mice with the MMTV-c-Neu mouse model. Methods: Fully congenic (100%) FVB/N-Tgfbr1+/- mice were crossed with FVB/N-Neu mice, and female virgin progeny were kept for analysis. Mammary glands were collected from 10, 12, and 40 week-old Neu and Neu;Tgfbr1+/- mice. Mice assessed for tumor development were sacrificed 80 days after the initial tumor palpation or at the earliest sign of morbidity. Whole lungs, tumor tissue, and primary tumor cells were collected for additional analysis. Long-term evaluation of 2-year-old Tgfbr1+/+ and Tgfbr1+/- mice was also conducted to directly assess the impact of Tgfbr1 haploinsufficiency on mammary gland and lung development. Results: Mammary gland whole mounts revealed that Neu;Tgfbr1+/- mice have more ductal branching at all time points compared to Neu mice. In the assessment of breast tumor development, Neu;Tgfbr1+/- mice were observed to have a significantly shorter tumor latency period (171 days) compared to Neu mice (220 days) (p=0.004). Seventy percent of Neu;Tgfbr1+/- mice (14/20) developed surface lung metastases, while 36.4% were observed in Neu mice (8/22), a borderline significant difference (p=0.061). The TGF-β-mediated growth inhibition of Neu;Tgfbr1+/- primary tumor cells was 32.2% lower than that of Neu tumor cells (p=0.007). Neu;Tgfbr1+/- tumor cells and tissue had significantly reduced Smad2/3 phosphorylation and total Cdkn1b (p27Kip1) expression compared to Neu mice. Long-term evaluation of Tgfbr1+/- and wild-type mice revealed no signs of mammary gland hyperplasia or differences in lung fibrosis in either group. Conclusion: This study is the first in vivo evidence that Tgfbr1 haploinsufficiency promotes breast carcinogenesis by increasing breast cancer proliferation. The relevance of this data to human breast cancer warrants additional investigations into the effects of decreased TGFBR1 signaling on tumor development and progression and identifies potential targets for prevention and treatment. Citation Format: Michael J. Pennison, Diana S. Rosman-Balzer, Lakisha Moore-Smith, Jacquelyn W. Zimmerman, Trenton R. Schoeb, Andra R. Frost, Ming Zhang, Peter M. Siegel, Boris C. Pasche. Tgfbr1 haploinsufficiency is a potent modifier of breast cancer risk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2013-1406 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 916A: Cancer cell proliferation is inhibited by specific modulation frequencies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Hepatocellular carcinoma (HCC), a leading cause of cancer death worldwide, now has the fastest growing rate in the United States. The intrabuccal administration of amplitude modulated electromagnetic fields (AM-EMF) is a novel, minimally invasive treatment modality. There is clinical evidence that this novel treatment approach elicits therapeutic responses in patients with cancer (1, 2). However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. Methods: Hepatocellular carcinoma cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumor-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 100 Hz to 21 kHz range as cancer-specific frequencies. Growth inhibition was evaluated by measuring tritiated thymidine incorporation. RNA-Seq was used to identify genes with differential expression in cells exposed to HCC-specific AM-EMF. Confocal laser scanning microscopy allowed the visualization of the mitotic spindle. Karyotype assessment compared HepG2 receiving HCC-specific AM-EMF to those not receiving exposure. Results: The growth of hepatocellular carcinoma and breast cancer cells was significantly decreased by hepatocellular carcinoma-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 hepatocellular carcinoma cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, hepatocellular carcinoma-specific modulation frequencies disrupted the mitotic spindle. Karyotype analysis was unrevealing. 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. Reference List (1) Barbault A, Costa F, Bottger B, Munden R, Bomholt F, Kuster N, et al. Amplitude-modulated electromagnetic fields for the treatment of cancer: Discovery of tumor-specific frequencies and assessment of a novel therapeutic approach. Journal of Experimental & Clinical Cancer Research 2009;28:51. (2) Costa FP, de Oliveira AC, Meirelles R, Machado MCC, Zanesco T, Surjan R, et al. Treatment of advanced hepatocellular carcinoma with very low levels of amplitude-modulated electromagnetic fields. Br J Cancer 2011;105:640-8. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 916A. doi:1538-7445.AM2012-916A

Abstract 4853: Constitutively decreased TGF-β signaling and risk for colorectal cancer

Background: Approximately one third of all colorectal cancers (CRC) are inherited, but less than 7% are attributable to known gene mutations. Germline variants within the TGF-β signaling pathway may account for colorectal cancer risk since TGF-β inhibits early tumor development through the activation of SMAD proteins. We have previously shown that Tgfbr1 haploinsufficiency is a potent modifier of colorectal cancer development in mice and humans. We have recently shown a significant association between a 3 SNP TGFBR1 haplotype and constitutively decreased TGFBR1 allelic expression, a phenotype found to occur frequently in patients with CRC. In this family-based study, we aimed at determining the association of TGFBR1 and SMAD7 with colorectal cancer risk. Methods: We studied 1,558 colorectal cancer cases and 2,640 healthy controls recruited by the Colorectal Cancer Family Registry (CCFR). We genotyped 14 TGFBR1 haplotype tagging SNPs as well as a SMAD7 SNP associated with CRC in prior GWA studies. SNPs were genotyped in all cases and controls using ABI9s TaqMan platform, and associations were determined using logistical regression analysis (significance set at p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4853. doi:1538-7445.AM2012-4853