Vimala Kaza

Genetic Exchange Between Homeologous Sequences in Mammalian Chromosomes Is Averted by Local Homology Requirements for Initiation and Resolution of Recombination

We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a “recipient” gene. Each substrate also contained one of several “donor” tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologous sequence sharing only 80% identity with the recipient gene. Mouse Ltk− fibroblasts were stably transfected with the various substrates and tk+ segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

A CCL8 gradient drives breast cancer cell dissemination.

The migration of cancer cells towards gradients of chemoattractive factors represents a potential, yet elusive, mechanism that may contribute to cancer cell dissemination. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells’ dissemination. In response to signals elicited by the neoplastic epithelium, CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. Manipulation of CCL8 activity influences the histology of the tumors and promotes major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. These findings exemplify how gradients of chemoattractive factors such as CCL8, drive metastasis and suggest that interference with their operation may provide means for breast cancer management.

Growth of human breast cancers in Peromyscus

ABSTRACT Modeling breast cancer in general and hormone-sensitive breast cancer, in particular in mice, has several limitations. These are related to the inbred nature of laboratory mice, and do not allow adequate appreciation of the contribution of the hosts genetic heterogeneity in tumor growth. In addition, the naturally low estrogen levels of mice makes estradiol supplementation obligatory for tumor growth. Here, we show that Peromyscus californicus, following cyclosporine-mediated immunosuppression, supports the growth of both MDA-MB-231 estrogen-independent and MCF7 estrogen receptor-positive breast cancers without exogenous estradiol supplementation. Tumor growth was inhibited by fulvestrant or letrozole, confirming that MCF7 xenografts remain hormone dependent in vivo and suggesting that P. californicus can be used as an alternative to conventional mice for the study of hormone-sensitive breast cancer. The fact that Peromyscus stocks are outbred also facilitates the study of breast cancer in genetically heterogenous populations. Summary: Outbred stocks of Peromyscus californicus (California mice), upon pharmacological immunosuppression, provide an alternative to conventional inbred mice models, and can support the growth of hormone-insensitive and hormone-sensitive human breast cancers.

The Value of Outbred Rodent Models in Cancer Research

Mouse models of breast cancer are valuable research tools, but their usefulness is restricted by a series of features inherent to their physiology, such as low endogenous estrogens and genetics (inbred status). Depending on the specific questions asked, outbred rodents like Peromyscus may provide answers that laboratory mice cannot.

Abstract 4896: Role of CDK8 in colon cancer hepatic metastasis

About half of all colon cancer patients will develop liver metastases and the 5-year survival for these patients is less than 13%, making colon cancer the second most lethal cancer worldwide. Cyclin dependent kinase 8 (CDK8), which regulates transcription but not cell cycle progression, has been identified as an oncogene amplified in many colon cancers. CDK8 acts as a positive mediator of oncogenic transcription pathways regulated by Wnt/beta-catenin and TGF-beta, both of which are strongly associated with tumor metastasis. In a colon cancer liver metastasis model based on splenic injection of CT26 murine colon carcinoma cells, treatment with Senexin B, a highly selective small-molecule inhibitor of CDK8 and its paralog CDK19, strongly inhibited metastatic growth in the liver and prolonged the survival of mice with hepatic metastases. In contrast to the effect on liver metastasis, CDK8 inhibition had little or no effect on cell growth in culture or at primary tumor sites. Hepatic metastasis was inhibited to the same extent when Senexin B was administered starting from the time of tumor injection or only during a later part of the study, suggesting that the drug affected metastatic growth in the liver rather than just initial colonization. Liver metastasis was similarly decreased by CDK8 knockdown in CT26 cells, suggesting that the anti-metastatic activity of Senexin B was due at least in part to its effect on tumor cells. Transcription profiling indicated that CDK8 inhibition by shRNA or Senexin B strongly decreased the expression of metastasis-associated metalloproteinases MMP 13, MMP10, and MMP3 and at the same time drastically increased the expression of TIMP3, a metalloproteinase inhibitor. TIMP3 overexpression in CT26 cells, like CDK8 inhibition, decreased hepatic metastasis. CDK8 inhibition also blocked TGF-beta or Wnt3a-stimulated transwell Matrigel invasion of CT26 cells in vitro. Knockdown of beta-catenin in CT26 cells decreased the expression of MMP3 and MMP13, while knockdown of SMAD4 (transcriptional mediator of TGF-beta pathway) induced TIMP3 expression. Beta-catenin knockdown decreased both the primary tumor growth in the spleen and metastatic growth in the liver, whereas SMAD4 knockdown, like that of CDK8, selectively inhibited liver metastasis. Senexin B also suppressed hepatic metastasis after splenic injection of human HCT116 colon carcinoma cells. In summary, our data identified CDK8 as a key transcriptional regulator of colon cancer metastatic growth in the liver, interacting with TGF-beta and Wnt/beta-catenin pathways and regulating the expression of MMPs and Timp3. CDK8/19 inhibitors, which are now entering clinical trials, may be effective for the treatment of hepatic metastasis of colon cancer. Citation Format: Jiaxin Liang, Mengqian Chen, Mythreye Karthikeyan, M. Marjorette Pena, Daniel Hughes, Vimala Kaza, Chang-Uk Lim, Eugenia Broude, Igor B. Roninson. Role of CDK8 in colon cancer hepatic metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2017-4896

Abstract A26: A self-sustained gradient of CCL8 drives breast cancer metastasis

The establishment of chemoattractive gradients between the neoplastic epithelium, the stroma and the peripheral tissues represents an attractive, yet elusive, mechanism that may contribute to metastasis. Here we provide evidence for the maintenance of a gradient of increasing CCL8 concentration between the epithelium, the stroma and the periphery that is instrumental for breast cancer cells dissemination promoting the directional movement of cancer cells from the site of primary tumor towards the periphery. Experiments in breast cancer cells cultured in vitro and in vivo studies in tumor-bearing mice showed that CCL8 production is enhanced in stromal fibroblasts at the tumor margins and in tissues at which breast cancer cells tend to metastasize such as the lungs and the brain. This stimulation of CCL8 expression in the stroma was driven by signals elicited by the neoplastic epithelium and could be recapitulated by administration of breast cancer cells conditioned media in tumor free mice. Experiments involving transwell-based migration assays established that CCL8-mediated chemoattraction was produced in breast cancer cells while genetic and antibody-based manipulation of CCL8 activity in mice influenced the histology of the tumors and promoted all major steps of the metastatic process such as invasion to adjacent stroma, intravasation and ultimately extravasation and seeding. Specifically, tumors growing in CCL8 deficient animals had well defined borders and high cellularity as opposed to the ill-defined margins of the tumors of wild type animals. In tumor-bearing mice acute i.v. administration of CCL8 increased the number of circulating tumor cells while antibody-mediated inhibition of CCL8 activity reduced the number of cancer cells invading towards adjacent fibroblasts that had been enclosed in matrigel and implanted proximally to the tumors. Only fibroblasts inoculated in the vicinity of breast tumors exhibited elevated CCL8 expression but not the fibroblasts growing in tumor-free mice, which is consistent with the active role of the epithelium in triggering CCL8 expression in the stroma. The stromal origin of CCL8 is also consistent with the strong correlation observed between circulating levels of CCL8 and tumor size, in EO771 mouse breast cancers at which CCL8 expression is minimal and in human MDA-MB-231 breast cancers at which species specific immunoassays permit the distinction between CCL8 originating from the neoplastic epithelium or the host. Analysis of publicly available data suggested that CCL8 is overexpressed in breast tumors as compared to normal breast tissue while high CCL8 expression in clinical breast cancers is associated with poor prognosis. These findings exemplify how self-sustained CCL8-like gradients of chemoattractive factors, between the epithelium, the stroma and the periphery can be induced by breast cancer cells to drive the metastatic process and suggest that interference with their operation may provide means for breast cancer management. Citation Format: Elena Farmaki, Ioulia Chatzistamou, Vimala Kaza, Hippokratis Kiaris. A self-sustained gradient of CCL8 drives breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A26.

Abstract PR08: Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19

Cyclin-dependent kinase 8 (CDK8), along with its closely related paralog CDK19, are transcription-regulating kinases that, unlike some other members of the CDK family, do not regulate cell cycle progression and are not required for the growth of normal cells or most of the tumor cell types. CDK8 has been identified as an oncogene that enhances the activity of several tumor-promoting transcriptional pathways (such as TGFβ, β-catenin, HIF1A and serum factors), mediating the elongation of transcription of newly activated genes. We have previously reported the discovery of the first selective small-molecule inhibitors of CDK8/19 and their ability to block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). We now conducted chemical optimization of the original inhibitors, yielding an optimized preclinical lead compound, Senexin B. Senexin B inhibits CDK8/19 in low nanomolar range in vitro and in vivo as an ATP pocket binder, with very high target selectivity as indicated by kinome profiling. It is highly water-soluble, bioavailable, and produces no limiting toxicity upon prolonged administration in mice, at doses that yield plasma concentrations exceeding cellular IC50 by 2-3 orders of magnitude. Senexin B has been tested for efficacy in several animal models addressing different aspects of tumor growth and progression. (i) Pretreatment of tumor-free mice with Senexin B significantly inhibited the growth of triple-negative breast cancer (TNBC) cells inoculated into mice subsequently to Senexin B administration, indicating a general chemopreventive effect on the normal tissue “soil”. (ii) Senexin B potentiated the tumor-suppressive effect of doxorubicin on established TNBC xenografts; this effect was associated with the suppression of NFκB-mediated transcriptional induction of tumor-promoting cytokines. (iii) Senexin B inhibited invasive growth into the muscle layer in an orthotopic xenograft model of MDA-MB-468 TNBC cells. (iv) In a spleen-to-liver colon cancer metastasis model of syngeneic mouse CT26 tumors, Senexin B treatment of mice had the same effect as CDK8 knockdown in tumor cells: suppression of metastatic growth in the liver without a significant effect on primary tumor growth in the spleen. Taken together, these results indicate that CDK8/19 inhibition produces chemopotentiating, chemopreventive and anti-metastatic effects in different types of cancer, inhibiting tumor progression by acting both at the tumor cells (the “seed”) and the tumor microenvironment (the “soil”) of cancers. Citation Format: Donald C. Porter, Mengqian Chen, Jiaxin Liang, Vimala Kaza, Alexander Chumanevich, Serena Altilia, Elena Farmaki, Marj Pena, Gary P. Schools, Ioulia Chatzistamou, Lawrence T. Friedhoff, Mark P. Wentland, Eugenia V. Broude, Hippokratis Kiaris, Igor B. Roninson. Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR08. doi:10.1158/1538-7445.CHTME14-PR08

Abstract 616: Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer

Most of castration-refractory prostate cancers (CRPC) continue to express androgen receptor (AR) that maintains its activity at low levels or even in the absence of androgen, driving tumor cell proliferation. Androgen-depleting drugs and androgen antagonists have little or no effect against AR+ cancers that are fully androgen-independent (AI). On the other hand, experimental compounds that cause AR degradation prevent AR-mediated repression of tumor-promoting genes, a tumor-suppressive effect of AR. We report a novel druggable target for CRPC, which mediates the induction but not the repression of gene expression by AR and inhibits the growth of AI prostate cancer (PCa) cells. This target is two closely related transcription-regulating kinases CDK8/CDK19 that unlike some other members of the CDK family are not required for cell cycle progression but play critical roles in several transcriptional signaling pathways. CDK8 and CDK19 are highly expressed in AR+ PCa cells and elevated under the conditions of androgen deprivation; CDK19 is overexpressed in AR+ PCa cell lines and in metastatic clinical PCa. A novel selective CDK8/19 kinase inhibitor (Senexin B) blocks ligand-induced AR-mediated transcriptional activation in androgen-dependent (AD) PCa cells but does not affect the AR-mediated gene repression. The CDK8/19 inhibitor also inhibits ligand-independent AR activation and AI cell growth in AI-PCa cells resistant to the anti-androgenic drug enzalutamide, both in vitro and in a mouse xenograft model. We have also discovered that CDK8/19 inhibition decreases NFκB mediated transcriptional activation of a group of tumor-promoting chemokines, such as CXCL1, CXCL2 and IL8, suggesting that CDK8/19 may promote prostate cancer chemoresistance and metastasis through the NFκB pathway. The above results suggest that CDK8/19 is an especially attractive therapeutic target for advanced PCa, which controls both AR-driven cell proliferation and other oncogenic pathways. Citation Format: Mengqian Chen, Vimala Kaza, Jiaxin Liang, Martina McDermott, Igor Roninson. Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate 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 616. doi:10.1158/1538-7445.AM2014-616

Abstract 4879: Targeting the seed and the soil of cancers with selective small-molecule inhibitors of CDK8/19: Chemopotentiating, chemopreventive, anti-invasive and anti-metastatic activities

Cyclin-dependent kinase 8 (CDK8), along with its closely related paralog CDK19, are transcription-regulating kinases that, unlike some other members of the CDK family, do not regulate cell cycle progression and are not required for the growth of normal cells or most of the tumor cell types. CDK8 has been identified as an oncogene that enhances the activity of several tumor-promoting transcription factors, mediating the elongation of transcription of newly activated genes. We have previously reported the discovery of the first selective small-molecule inhibitors of CDK8/19 and their ability to block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). We now conducted chemical optimization of the original inhibitors, yielding an optimized preclinical lead compound, Senexin B. Senexin B inhibits CDK8/19 in low nanomolar range in vitro and in vivo as an ATP pocket binder, with very high target selectivity as indicated by kinome profiling. It is highly water-soluble, bioavailable, and produces no limiting toxicity upon prolonged administration in mice, at doses that yield plasma concentrations exceeding cellular IC50 by 2-3 orders of magnitude. Senexin B has been tested for efficacy in several animal models addressing different aspects of tumor growth and progression. (i) Pretreatment of tumor-free mice with Senexin B significantly inhibited the growth of triple-negative breast cancer (TNBC) cells inoculated into mice subsequently to Senexin B administration, indicating a general chemopreventive effect on the normal tissue “soil”. (ii) Senexin B potentiated the tumor-suppressive effect of doxorubicin on established TNBC xenografts. (iii) Senexin B inhibited invasive growth into the muscle layer in an orthotopic xenograft model of MDA-MB-468 TNBC cells. (iv) In a spleen-to-liver metastasis model of syngeneic mouse CT26 tumors, Senexin B treatment of mice had the same effect as CDK8 knockdown in tumor cells: suppression of metastatic growth in the liver without a significant effect on primary tumor growth in the spleen. Taken together, these results indicate that CDK8/19 inhibition produces chemopotentiating, chemopreventive and anti-metastatic effects in different types of cancer, inhibiting tumor progression at the organismal level. Citation Format: Donald C. Porter, Jiaxin Liang, Vimala Kaza, Alexander A. Chumanevich, Serena Altilia, Elena Farmaki, Mengqian Chen, Gary P. Schools, Ioulia Chatzistamou, Marj M. Pena, Lawrence T. Friedhoff, Mark P. Wentland, Eugenia Broude, Hippokratis Kiaris, Igor B. Roninson. Targeting the seed and the soil of cancers with selective small-molecule inhibitors of CDK8/19: Chemopotentiating, chemopreventive, anti-invasive and anti-metastatic activities. [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 4879. doi:10.1158/1538-7445.AM2014-4879