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Supplementary Figure-1. Inhibitory concentration 50 of curcumin and AKBA.

Supplementary Figure-8. Comparative gene-expression analysis of FBXW7, cMyc, CDK4, CDK6, Cyclin E1 and Cyclin D1 from xenograft tumors assessed by qRT-PCR.

Colorectal cancer is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are well-established dietary botanicals with potent antitumorigenic properties that have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may, in part, be mediated through regulation of key cancer-related microRNAs (miRNA) and their downstream gene targets. Here, we investigated the antitumorigenic effects of curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in colorectal cancer cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell-cycle arrest in colorectal cancer cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways, including key cell-cycle regulatory genes. Combined bioinformatics and in silico analysis identified apoptosis, proliferation, and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anticancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in colorectal cancer cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein, we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer. Cancer Prev Res; 8(5); 431–43. ©2015 AACR.

Supplementary Figure-7. The effects of miR-27a knockdown on cell function in colorectal cancer cells.

Supplementary Figure-4. The effects of curcumin and AKBA on miR-34a, miR-27a, miR-16 and miR-145 expression in CRC cell lines.

Supplementary Figure-5. Curcumin and AKBA modulate genes involved in cell cycle regulation.

Supplementary Figure-2. Curcumin and AKBA exert cytotoxicity and suppress colony formation on CRC cell lines.

Supplementary tables and Figure Legends

Supplementary Figure-3. Curcumin and AKBA inhibit cellular proliferation in 3D cultures.

Supplementary Figure-6. The effects of miR-34a over-expression on cell function colorectal cancer cells.

Colorectal cancer is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Both curcumin and boswellic acids are well-established dietary botanicals with potent antitumorigenic properties that have been shown to modulate multiple oncogenic pathways. Recent data suggest that the chemopreventive effects of these botanicals may, in part, be mediated through regulation of key cancer-related microRNAs (miRNA) and their downstream gene targets. Here, we investigated the antitumorigenic effects of curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) on modulation of specific cancer-related miRNAs in colorectal cancer cells and validated their protective effects in vivo using a xenograft mouse model. Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell-cycle arrest in colorectal cancer cell lines, and these effects were significantly enhanced with combined treatment. Gene-expression arrays revealed that curcumin and AKBA regulated distinct cancer signaling pathways, including key cell-cycle regulatory genes. Combined bioinformatics and in silico analysis identified apoptosis, proliferation, and cell-cycle regulatory signaling pathways as key modulators of curcumin and AKBA-induced anticancer effects. We discovered that curcumin and AKBA induced upregulation of tumor-suppressive miR-34a and downregulation of miR-27a in colorectal cancer cells. Furthermore, we demonstrated in a mouse xenograft model that both curcumin and AKBA treatments suppressed tumor growth, which corresponded with alterations in the expression of miR-34a and miR-27a, consistent with our in vitro findings. Herein, we provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in colorectal cancer. Cancer Prev Res; 8(5); 431–43. ©2015 AACR.

Types of T-cell responses are categorized on the basis of a limited number of molecular markers selected usinga prioriknowledge about T-cell immunobiology. We sought to develop a novel systems-based approach for the creation of an unbiased framework enabling assessment of antigenic-peptide specific T-cell responsesin vitro. A meta-analysis of transcriptome data from PBMCs stimulated with a wide range of peptides identified patterns of gene regulation that provided an unbiased classification of types of antigen-specific responses. Further analysis yielded new insight about the molecular processes engaged following antigenic stimulation. This led for instance to the identification of transcription factors not previously studied in the context of T-cell differentiation. Taken together this profiling approach can serve as a basis for the unbiased characterization of antigen-specific responses and as a foundation for the development of novel systems-based immune profiling assays.

To enhance the therapeutic index of allogeneic hematopoietic stem cell transplantation (HSCT), we immunized ten HLA-matched sibling donors prior to stem cell collection with recipient-derived clonal myeloma immunoglobulin, idiotype (Id), as a tumor antigen, conjugated with keyhole limpet hemocyanin (KLH). Vaccinations were safe in donors and recipients. Donor-derived KLH- and Id-specific humoral and central and effector memory T cell responses were detectable by day 30 after HSCT and were boosted by post-transplant vaccinations at 3 months in most recipients. One patient died prior to booster vaccinations. Specifically, after completing treatment 8/9 myeloma recipients had persistent Id-specific immune responses and 5/9 had improvement in disease status. Although regulatory T cells increased after vaccination, they did not impact immune responses. At a median potential follow-up period of 74 months, six patients are alive, the 10 patients have a median progression-free survival of 28.5 months, and median overall survival has not been reached. Our results provide proof of principle that neoantigen and tumor antigen-specific humoral and cellular immunity could be safely induced in HSCT donors and passively transferred to recipients. This general strategy may be used to reduce relapse of malignancies and augment protection against infections after allogeneic HSCT.

Purpose: Colorectal cancer (CRC) is one of the most common causes of cancer-associated mortality worldwide, but it is truly a preventable disease. Accumulating evidence indicates that several dietary botanicals, including curcumin and boswellic acids, have potent anti-inflammatory and anti-tumorigenic properties. However, the molecular mechanisms underlying the anti-cancer potential of these compounds remain poorly understood. More recent data suggest that the chemopreventive effects of these botanicals may in part be mediated through regulation of key cancer-related microRNAs (miRNAs), and their downstream gene targets. This study aimed to determine whether curcumin and 3 acetyl-11-keto-β-boswellic acid (AKBA) exert their anti-tumorigenic effects through modulation of specific miRNAs in CRC cell lines and a xenograft mouse model. Experimental Design: We first determined the effects of curcumin and AKBA on cellular proliferation, apoptosis and cell cycle dynamics in a panel of CRC cell lines. Next, we used Illumina's Human HT-12 V4 array gene expression arrays to systematically identify specific gene signaling pathways regulated by curcumin and AKBA. Subsequently we used combined bioinformatics and in-silico approaches to identify miRNAs and molecular pathways activated by curcumin and AKBA individually, and their combination. We determined the functional role of curcumin and AKBA-related miRNAs in CRC cell lines, and validated these findings for the chemopreventive efficacy of curcumin and AKBA in a xenograft animal model. Results: Both curcumin and AKBA inhibited cellular proliferation, induced apoptosis and cell cycle arrests in all CRC cell lines, and these effects were significantly enhanced with combined vis-à-vis individual treatments with the botanicals. Gene-expression arrays revealed that curcumin and AKBA regulated key cell cycle controlling genes including: cMYC, CCND1, CCNE1 and CDK4. Combined bioinformatics and in-silico analysis identified apoptosis, proliferation and cell cycle regulatory signaling pathways as the key modulators of curcumin and AKBA-induced anti-cancer effects, and further discovered miR-27a and miR-34a as the most significantly differentially expressed miRNAs following treatment with the two botanicals. In support of our in vitro findings, mouse xenograft experiments further confirmed that both curcumin and AKBA suppressed tumor size and volume in mice, and that these effects were orchestrated through modulation of miR-27a and miR-34a expression in the animal model. Conclusion: We provide novel mechanistic evidence for the chemopreventive effects of curcumin and AKBA through regulation of specific miRNAs in CRC. The enhanced anti-tumorigenic effect by combined treatment with these botanicals provides a potential rationale for the use of such diet-based botanicals for the chemoprevention of this malignancy. Citation Format: Shusuke Toden, Yoshinaga Okugawa, Keun Hur, Thomas A. Jascur, Constanze Burhrmann, Durgha Nattamai, Esperanza Anguiano, Mehdi Shakibaei, C. Richard Boland, Ajay Goel. Novel evidence for chemopreventive effects of curcumin and boswellic acid through regulation of mir-27a and mir-34a in human colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4119. doi:10.1158/1538-7445.AM2014-4119

Follicular lymphoma (FL) is one of the most immune responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of endogenous immunity as well as therapeutic vaccines and adoptive T-cell therapy. CD4+CD25+CD127loFoxp3+ regulatory T cells (Tregs) are one of the most potent suppressors of effector T cells and were recently shown to be increased in peripheral blood and tumors of patients with various malignancies. Here, we determined the percentage of Tregs, in the tumors and the peripheral blood mononuclear cells (PBMC) of patients with FL (n = 13) at the time of initial diagnosis and after induction of clinical remission with a uniform cyclophosphamide and doxorubicin containing chemotherapy regimen, PACE (Modified ProMACE without Methotrexate; Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide). We observed that Tregs are markedly increased in number both in the tumors (median 19.45%, range 13.4%–44.5%) and PBMC (median 17.7%, range 7.9%–64.5%) of patients with FL at the time of initial diagnosis as compared with PBMC from normal donors (median 3.33%, range 1.88%–6.3%). Unexpectedly, Tregs persisted in high numbers in peripheral blood after induction of clinical remission with a cyclophosphamide and doxorubicin containing chemotherapy regimen (median 19.95%, range 7.6%–67.2%). To determine the immunosuppressive effects of Tregs, we assessed the proliferation of CFSE-labeled effector T cells following polyclonal stimulation with anti-CD3 and anti-CD28 antibodies. The presence of high numbers of Tregs was associated with significantly decreased proliferation of peripheral blood CD4+ T cells in patients with FL both at baseline (median 24.9%, range 3.7%–40.7%) and after chemotherapy induced clinical remission (median 15.8%, range 1.5%–23.5%) as compared with normal donors (median 38%, range 11.7%–58.8%). A similar decrease in proliferation was also noted in CD8+ T cells in the presence of high numbers of Tregs both at baseline and after induction of clinical remission in patients with FL as compared with normal donors. More importantly, intratumoral and peripheral blood Tregs significantly inhibited effector T cells proliferating in response to autologous tumor cell stimulation. In conclusion, these results suggest that CD4+CD25+CD127loFoxp3+ Tregs that inhibit the function of tumor-specific effector T cells are markedly increased in number in both tumor and peripheral blood of patients with FL and persist in high numbers despite induction of clinical remission with standard cyclophosphamide and doxorubicin containing chemotherapy. This observation may potentially explain the failure of two recent Phase III therapeutic vaccine trials in patients with FL. Furthermore, these results suggest that a state of peripheral tolerance continues to be present after induction of clinical remission and strategies that deplete or block function of Tregs may be necessary to enhance the efficacy of therapeutic vaccines and adoptive T-cell therapy in patients with follicular and possibly other non-Hodgkin’s lymphomas.

Follicular lymphoma is one of the most immune-responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of immunotherapies such as vaccines and adoptive T-cell therapy. The inhibitory receptor programmed death 1 (PD1), a negative regulator of activated T cells was recently shown to be upregulated on the surface of HIV-specific CD4+ and CD8+ T cells in humans and was associated with impaired T-cell function. Blockade of the immunoregulatory PD-1/PD-ligand 1 (PD-L1) pathway with antibodies against the PD-L1 augmented the function of HIV-specific CD4+ and CD8+ T cells (Day CL et al, Nature, 2006). To investigate the role of PD-1 in lymphoma, we examined PD-1 expression on peripheral blood mononuclear cells (PBMC) and intratumoral T cells in patients with follicular lymphoma prior to therapy. We observed that PD-1 expression is significantly upregulated on peripheral blood and intratumoral CD4+ and CD8+ T cells in patients with follicular lymphoma as compared with normal donor PBMC. Furthermore, PD-1 expression was significantly higher on intratumoral (mean 61%, range 34% to 86%) compared with peripheral blood CD4+ T cells (mean 25%, range 9 to 40%). Likewise, PD-1 expression was significantly higher on intratumoral (mean 44%, range 31% to 69%) compared with peripheral blood CD8+ T cells (mean 16%, range 9 to 31%). PD-1 expression on CD4+ and CD8+ T cells was associated with impaired type 1 cytokine production (IL-2, TNFa, and IFNg) and blockade of the PD-1/PD-ligand pathway with antibodies against PD-1 significantly enhanced T-cell function. These data indicate that the immunoregulatory PD-1/PD-ligand pathway is operative in patients with follicular lymphoma. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T-cells in follicular lymphoma in combination with other immunomodulatory strategies such as vaccines and adoptive T-cell therapy.