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Background: The taxanes and platinum agents provide substantial improvement in the treatment of breast and other cancers. Neuropathy is often the dose limiting toxicity of these agents and can devastatingly affect the patient due to diminished fine motor skills and pain in the hands and feet that diminishes the ability to exercise and interact in normal life. Clinically significant neuropathy occurs in 40% of patients and >10% will persist past a year causing permanent effects on quality of life. Perhaps more devastating, patients often have to discontinue effective treatment due to the development of these symptoms. Additionally, anemia contributes to dose reductions and delays that compromise therapy and cause significant fatigue that diminishes quality of life. Interventions that could reduce these symptoms would provide a substantial improvement in the ability to care for these patients. Toward this end we have developed a non-toxic dietary approach that incorporates supplementation with several natural products. Together, this therapeutic approach, called CS.001 is able to alleviate many of the negative side effects of breast cancer chemotherapy. Methods: C57BL/6 mice were treated with Paclitaxel weekly or Oxaliplatin three times weekly. Animals were tested for neuropathic pain using a cold sensitivity test [acetone test] and sensitivity to mechanical stimulus [Von Frey Test]. A CBC and CMP were performed to assess systemic toxicity. Animals were provided with a nutritional complete diet that limited their carbohydrates to 10% of total caloric input and were additionally supplemented with the following natural products: [1] Medium chain triglycerides [30g/kg], [2] Curcumin [1200mg/kg], [3]EGCG [1200mg/kg] and [4]broccoli sprout powder [20g/kg]. Animals were placed on the CS.001 diet 1 week before beginning chemotherapy. Results: Paclitaxel & Oxaliplatin treatment resulted in a statistically significantly increase in sensitivity to cold stimulus [Acetone test, p Conclusion: Dietary intervention combined with a supplementation of 4 natural products was able to attenuate chemotherapy induced anemia and neuropathy following chemotherapy treatment in mice. This combination is a promising quality of life intervention to evaluate in cancer patients receiving taxanes or platinums. Citation Format: Coy D Heldermon, Ben G Griffith, Matt Bloom, Jennifer Owen, Regina J Martuscello, Yosef Schwartz, Brent A Reynolds, Loic P Deleyrolle. A combination natural product therapy attenuates common side effects associated with chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-15-10.

Specific treatment options for patients with metastatic breast cancers, especially of the triple negative subtype (TNBC), are limited and most patients develop resistance to radiation and/or chemotherapy. Because of this, the mortality rate in women with TNBC remains high. Carbonic anhydrases (CAs), most specifically membrane-bound CAIX, has been shown to play an important role in tumor progression and as a result is validated as a therapeutic target in several aggressive cancers. In breast cancer, CAIX expression has been shown to be ubiquitous in hypoxic TNBC tissues and is considered a prognostic marker and a potential therapeutic target. Therefore targeting CAIX activity, using small molecule inhibitors, in these highly aggressive and metastatic TNBCs may serve to improve overall disease free survival and therapeutic outcome in clinics. Our goal for this study was to 1) Identify and/or develop a cell line that expresses CAIX in a hypoxia independent manner and is representative of TNBCs and 2) To target CAIX activity in this cell line and observe the effects of CAIX inhibition on its growth and metastasis. Our hypothesis is that CAIX inhibition, in the context of a hypoxic and/or acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. To achieve our aims, we newly developed and characterized an endogenous CAIX-expressing cell line named UFH-001. Our results show that CAIX expression and activity in this cell line, although endogenous under normoxic conditions, is increased more than 2 fold under hypoxic conditions. This cell line is also representative of TNBCs (ER/PR and HER2 negative) with a more epithelial-like morphology and phenotype. UFH-001 cells grow aggressively and have the ability to migrate/invade and form tumors in vivo. In these studies we have also shown how inhibition of CAIX activity, using sulfonamide-based inhibitors, affects UFH-001 growth in an apoptosis independent manner and decrease the migratory/inhibitory capacity of these cells. Taken together, our observations indicate that CAIX is a viable small molecular drug target for the treatment of patients with TNBCs. Citation Format: Mam Y. Mboge, Zhijuan Chen, Lingbao Ai, Chingkuang Tu, Fabrizio Carta, Claudiu Supuran, Christopher J. Frost, Zaihui Zhang, Robert McKenna, Coy Heldermon, Susan C. Frost. UFH-001: A novel triple-negative, CAIX-positive cell line for small-molecule targeting of human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2102.

EGFR and/or HER2 are frequently overexpressed oncoproteins in HER2+ breast cancer, Triple Negative Breast Cancer (TNBC), and Inflammatory Breast Cancer (IBC). However, EGFR and/or HER2 targeting agents have failed to make major advances in improving survival for these cancer patients, largely because these cancers frequently develop resistance to these drugs. The identification of novel treatment strategies is critically needed. Overexpression of EGFR and/or HER2 presents a severe burden to the secretary pathway, as HER-family proteins EGFR, HER2, HER3 share conserved extracellular cysteine-rich and proline-rich repeats that form numerous disulfide bonds and control cis-trans isomerization in the protein folding processes. We demonstrated that the disulfide bond disrupting Agents (DDAs) selectively killed EGFR+ and/or HER2+ breast cancer cells in vitro and decreased tumor growth of HER2+ cancer in a xenograft mouse model. DDA-dependent anti-cancer actions resulted from downregulation of EGFR/HER2/HER3, induction of AKT dephosphorylation, and activation of Endoplasmic Reticulum (ER) stress. We also investigated a novel proteotoxic combination therapy employing DDAs and Proline Isomerase Inhibitors (PIIs) in treating breast cancers. PIIs potentiated the efficacy of DDAs in downregulating EGFR/HER2/HER3 levels and initiating ER stress, and the combination was highly synergistic in reducing EGFR+ and/or HER2+ breast cancer cell viability. Importantly, the combination of DDAs and PIIs overcame single drug resistance, significantly decreased tumor volumes and increased the survival of xenograft mice. Evidence that the drug combination was effective against metastatic breast cancer cells is also provided. Citation Format: Mengxiong Wang, Renan Ferreira, Mary Law, Elham Yaaghubi, Coy Heldermon, Ronald Castellano, Brian Law. A novel proteotoxic combination therapy for EGFR+ and HER2+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 447.

Unmet needs in the breast cancer field include the development of effective treatments for 1) drug-resistant HER2+ breast cancers, 2) Triple-Negative (HER2-, ER-, PR-) Breast Cancers (TNBCs), and 3) Inflammatory Breast Cancers (IBCs). Treatment-refractory HER2+ tumors retain HER2 expression after acquiring drug resistance, and large fractions of TNBCs and IBCs overexpress either HER2 or EGFR. Thus, drugs that selectively kill breast tumors that overexpress EGFR or HER2 could benefit patients suffering from these three classes of treatment-refractory tumors. Our team has identified a class of compounds termed Disulfide bond Disrupting Agents (DDAs) that selectively kill cancer cells that overexpress HER2 or EGFR. DDA-mediated cancer-selective cytotoxicity results from A) downregulation of the EGFR/HER2/HER3 proteins, B) inactivation of the Akt oncogene, and C) activation of the Unfolded Protein Response (UPR). Importantly DDAs only elicit these responses in cancer cells that overexpress EGFR or HER2. DDAs exhibit striking anticancer activity in human HER2+ xenograft tumors grown orthotopically in mice, with no evidence of toxicity. DDAs function through mechanisms distinct from those of HER2 specific monoclonal antibodies and tyrosine kinase inhibitors, and biochemical studies indicate that DDAs are complementary with Lapatinib with respect to blockade of HER2 driven proliferative and survival signaling. Current efforts are directed toward identifying mechanism-based drugs that synergize with DDAs to potentiate HER1-3 downregulation, Akt inhibition, and UPR, and to kill HER2+/EGFR+ patient-derived xenograft tumors that are refractory to current therapies. Citation Format: Brian K. Law, Mary E. Law, Renan B. Ferreira, Mengxiong Wang, Bradley J. Davis, Ronald K. Castellano, Coy D. Heldermon. Multifaceted targeting of drug-resistant EGFR+ and HER2+ breast tumors [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 4050. doi:10.1158/1538-7445.AM2017-4050

Tumor microenvironment substantially influences the process of tumorigenesis. Extracellular acidification within the tumor microenvironment is an indicator of an aggressive cancer and a marker for poor patient outcome. In solid tumors, hypoxia leads to extracellular acidosis. Carbonic anhydrases (CA) are thought to regulate intracellular and extracellular pH (pHi and pHe, respectively). To explore the effect of CAs in breast cancer, we compared the expression and activity of two membrane bound CAs, CAIX and CAXII, between triple negative breast cancer cells (TNBCs) and luminal breast cancer cells (LBCs). We chose five different TNBC and LBC lines. Our data show that, among the TNBC lines, CAIX expression increased in three of the five lines: HBL100, SUM159, and the new UFH-001 cells under hypoxic condition. UFH-001 cells also showed strong constitutive expression. None of these TNBC lines expressed CAXII or estrogen receptor (ER). In LBC lines, four of the five lines constitutively expressed CAXII: T47D, MCF7, SKBR and SUM52 cells. CAXII expression was not hypoxia-dependent. Each of the five luminal lines expressed ER. We also examined CA expression in a tumor graft model. In tumors grown from cells derived from TNBC patients, we observed CAIX expression in four of six sample sets. In tumors derived from ER-positive LBC patients, all five expressed CAXII. We also used the 18O exchange method to assess CA activity. Two TNBC lines: UFH-001 and HBL100 cells showed that CAIX activity increased in hypoxic conditions which was blocked by an impermeant sulfonamide CA inhibitor (N3500). In the luminal lines, we detected CAXII activity in T47D and MCF7 cells that was also inhibited by N3500. Like CAXII protein expression in these cells, CAXII activity was not affected by hypoxia. We also evaluated the effect of pH on CA activity in TNBC and LBC lines. Both CAIX and CAXII showed increased activity in response to reduced pH, which is expected in a bicarbonate-based system. However, UFH-001 cells also exhibited a hypoxic-dependent increase in CAIX activity which is associated with increased protein expression. In conclusion, these observations demonstrate that CAIX expression is associated with the TNBC phenotype. Based on our activity data, we would predict that CA activity in TNBC tumors will be sensitive to both hypoxia (based on enhanced expression) and reduced pH. This change in activity may serve to regulate pH in the tumor microenvironment favoring an aggressive phenotype. On the other hand, LBC tumors, which are ER-positive, are only associated with CAXII expression. In luminal cells, we expect that only pH and not hypoxia will affect CAXII activity. This may, in part, explain the more positive prognosis in patients with CAXII expression. Note: This abstract was not presented at the meeting. Citation Format: Zhijuan Chen, Mam Y. Mboge, Chingkuang Tu, Lingbao Ai, Coy Heldermon, Susan C. Frost. Comparison of carbonic anhydrase & activity between triple-negative & luminal breast cancer cells [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 5936. doi:10.1158/1538-7445.AM2017-5936