Key Proliferative Pathways Research Articles

Abstract 3164: Disulfiram re-sensitizes ER+ breast cancer cells resistant to chemotherapy through an evolutionary double-bind

Abstract Most ER+ metastatic breast cancer patients develop resistance to endocrine therapy and are also typically cross-resistant to chemotherapy, resulting in disease progression. However, chemotherapy resistant lines have been shown to acquire cross-sensitivity to therapies such as disulfiram, a drug used to treat alcoholism. Because of this cross-sensitivity, we hypothesized the existence of an “evolutionary double-bind”, a situation where treatment with one drug improves the response to a different agent. This can occur when resistance mechanisms rewire key proliferative pathways, creating potentially targetable vulnerabilities to be exploited. While recent studies have applied theoretical ecological and evolutionary principles in understanding cancer progression, it remains a new and exciting endeavor to quantify effective evolutionary games in refractory cancer to design alternative treatment strategies that induce an evolutionary double-bind. Here, we developed ER+ breast cancer cell lines sensitive and resistant to paclitaxel or doxorubicin to test the cross-sensitivity and efficacy of chemotherapy and disulfiram as an alternative treatment strategy. To quantify this cross-sensitivity of chemotherapy and disulfiram and use the double-bind to mitigate resistance, we also developed a game-theoretic mathematical modeling framework with parametrization performed using the “evolutionary game assay” technique, a direct measurement of payoff matrices that specify evolutionary games measured in cancer. Spheroids of varied initial frequencies of sensitive and resistant cells (100% monoculture; 75/25, 50/50, and 25/75 sensitive and resistant coculture) were treated with chemotherapy and disulfiram. The mathematical model was parameterized from the in vitro experimental data and as expected, treating with chemotherapy selects for resistant cells. However, disulfiram alone shifts the selection dynamics to favor sensitive cells. These modeling results predict the existence of a double-bind between chemotherapy and disulfiram where disulfiram selects for the sensitive subpopulation, which then re-sensitizes the spheroids to subsequent administration of chemotherapy. Importantly, the model predicts that the double-bind is non-symmetric and does not exist when treatments are given in combination. These predictions suggest the importance of timing/dosing strategies in which several schemes were further validated in vitro, confirming the superior efficacy of chemotherapy followed by disulfiram in blocking resistance emergence. This project integrates experimental data with mathematical modeling to identify a treatment double-bind in ER+ breast cancer. The integrative experimental-mathematical framework developed here has broad applicability in designing evolution-based or adaptive therapy treatment strategies across a wide range of cancer types. Citation Format: Rena Emond, Jeffrey West, Alexander Anderson, Andrea H. Bild. Disulfiram re-sensitizes ER+ breast cancer cells resistant to chemotherapy through an evolutionary double-bind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3164.

Sexual Dimorphism in Colon Cancer.

A higher incidence of colorectal cancer (CRC) is found in males compared to females. Young women (18–44 years) with CRC have a better survival outcome compared to men of the same age or compared to older women (over 50 years), indicating a global incidence of sexual dimorphism in CRC rates and survival. This suggests a protective role for the sex steroid hormone estrogen in CRC development. Key proliferative pathways in CRC tumorigenesis exhibit sexual dimorphism, which confer better survival in females through estrogen regulated genes and cell signaling. Estrogen regulates the activity of a class of Kv channels (KCNQ1:KCNE3), which control fundamental ion transport functions of the colon and epithelial mesenchymal transition through bi-directional interactions with the Wnt/β-catenin signalling pathway. Estrogen also modulates CRC proliferative responses in hypoxia via the novel membrane estrogen receptor GPER and HIF1A and VEGF signaling. Here we critically review recent clinical and molecular insights into sexual dimorphism of CRC biology modulated by the tumor microenvironment, estrogen, Wnt/β-catenin signalling, ion channels, and X-linked genes.

Hedgehog acyltransferase as a target in pancreatic ductal adenocarcinoma.

Sonic Hedgehog (Shh) is abnormally expressed in pancreatic cancer and is associated with disease onset and progression. Inhibition of Shh signaling is thus an attractive clinical target for therapeutic intervention. Most efforts to block Shh signaling have focused on inhibitors of Smoothened, which target the canonical Shh signaling pathway. These approaches have met with limited success, in part due to development of resistance-conferring mutations and contributions from non-canonical signaling pathways. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for the attachment of palmitate onto Shh, is a novel target for inhibition of Shh signaling in pancreatic cancer cells. Depletion of Hhat with lentivirally delivered small hairpin RNA decreased both anchorage-dependent and independent proliferation of human pancreatic cancer cells. In vivo, Hhat knockdown led to reduction of tumor growth in a mouse xenograft model of pancreatic cancer. RU-SKI 43, a small molecule inhibitor of Hhat recently developed by our group, reduced pancreatic cancer cell proliferation and Gli-1 activation through Smoothened-independent non-canonical signaling. In addition, RU-SKI 43 treatment inhibited two key proliferative pathways regulated by Akt and mTOR. This work demonstrates that Hhat has a critical role in pancreatic cancer and that a small molecule inhibitor of Hhat can successfully block pancreatic cancer cell proliferation. It also highlights the importance of developing optimized Hhat inhibitors to be used as therapeutics in pancreatic cancer, as well as in other malignancies characterized by Shh overexpression.

Targeting cell cycle and hormone receptor pathways in cancer

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

Osteoblast-Stem Cell Contact Is Required for B-Lymphopoietic Proliferation, Not Differentiation.

Abstract 3635Poster Board III-571Nonparenchymal stromal cells form the cellular constituents of tissue-specific stem cell niches. One key question in stem cell biology is the nature of signals provided by stromal cells in general, and via stem cell-stromal cell contact in particular, that direct the survival, proliferation and differentiation of tissue stem cells. We and others have previously reported that osteoblasts dictate the differentiation of hematopoietic stem cells (HSC) to B lymphopoiesis, supporting the acquisition of E2A, EBF1 and Pax5, as well as cell surface B220, CD18 and sIgM, with Flt3-ligand (FL) and interleukin 7 (IL-7) required for these activities. In this context, a fundamental unanswered question is whether, and if so why, B lymphopoiesis requires direct cell-cell contact between HSCs and osteoblasts. In the present study, we asked whether HSC contact with osteoblasts plays its principle role in B lymphopoiesis by supporting B differentiation, by supported proliferation, or both. Magnetically purified mouse Lin- Kit+ (LK) bone marrow cells were cultured for 11 days in the presence of 50 ng/ml FL plus 100 ng/ml IL-7, in direct contact with OP-9 or MS-5 osteoblastoid cells, or in proximity to OP-9/MS-5 cells but separated by a Transwell filter. Following co-culture, the hematopoietic cells were counted, the presence of B lineage-specific cell surface markers measured by flow cytometry, and the expression levels of B-lineage transcription factors E2A, EBF and Pax5 measured by quantitative PCR. LK cultured in FL + IL-7 acquired B220 as well as low levels of E2A expression, but did not express Pax5 expression nor acquire cell surface CD19 or IgM, suggesting differentiation to Pre-Pro B cells but not beyond; in contrast, LK cultured in direct contact with OP-9 differentiated fully to mature B cells, expressing Pax5 and cell surface CD19 and IgM. LK cultured above OP9 cells but with cell-cell contact prevented by an interposed Transwell also fully acquired the transcription factor and cell surface antigenic profiles of mature B cells, but the proliferation that accompanies OB-contact driven differentiation was blocked:Cells/mL Day 0Cells/mL Day 11Percent of InputOP-9 Direct Contact2.3 × 1057.8 × 1063400%MS-5 Direct Contact2.3 × 1051.6 × 106670%OP-9 Transwell2.3 × 1051.3 × 1045%MS-5 Transwell2.3 × 1052.0 × 1049%These data indicate that all the signals required to induce HSC differentiation to mature B cells are contained within the combination of cytokines and other molecules secreted by OP-9 cells, but that direct contact with OP-9 cells provides a proliferative stimulus not achieved by OP-9 secreted molecules, either via a direct receptor-ligand interaction or indirectly, by contact-induced secretion of specific soluble proliferative factors from osteoblasts. Experiments to distinguish these alternatives, and to identify whether such signals trigger nutrient influx, direct activation of Akt, MAPK or other key proliferative pathways, are now in progress. The proliferative role of stromal cell-parenchymal cell contact within the stem cell niche likely may have general implications to normal and neoplastic cell physiology. Disclosures:No relevant conflicts of interest to declare.

Uncovering Pandora’s vase: the growing problem of new toxicities from novel anticancer agents. The case of sorafenib and sunitinib

As newer, molecularly targeted, anticancer drugs are entering clinical practice, a wide array of previously unrecognised and ill defined side effects of these drugs are increasingly observed. Sorafenib and sunitinib are two of these novel agents, acting on tumour angiogenesis as well as on other key proliferative pathways; recently approved for the treatment of advanced kidney cancer, they may cause peculiar cutaneous, vascular and mucosal toxicities, including hand-foot skin reaction, skin rash, hypertension and GERD-like oesophagitis/gastritis. In this review, we shall deal with these poorly recognised, but sometimes extremely distressing, toxicities; pathophysiologic mechanisms will be discussed and suggestions for treatment of each toxicity will be proposed, based on the few pieces of evidence available and, especially, on our empirical experience.