Purpose The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improve efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Methods Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production was analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. Results We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. Conclusions We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.

Human papillomavirus induced (HPV+) cancer incidence is rapidly rising, comprising 60–80% of oropharyngeal squamous cell carcinomas (OPSCCs); while rare, recurrent/metastatic disease accounts for nearly all related deaths. An in vivo pre-clinical model for these invasive cancers is necessary for testing new therapies. We characterize an immune competent recurrent/metastatic HPV+ murine model of OPSSC which consists of four lung metastatic (MLM) cell lines isolated from an animal with HPV+ OPSCC that failed cisplatin/radiation treatment. These individual metastatic clonal cell lines were tested to verify their origin (parental transgene expression and define their physiological properties: proliferation, metastatic potential, heterogeneity and sensitivity/resistance to cisplatin and radiation. All MLMs retain expression of parental HPV16 E6 and E7 and degrade P53 yet are heterogeneous from one another and from the parental cell line as defined by Illumina expression microarray. Consistent with this, reverse phase protein array defines differences in protein expression/activation between MLMs as well as the parental line. While in vitro growth rates of MLMs are slower than the parental line, in vivo growth of MLM clones is greatly enhanced. Moreover, in vivo resistance to standard therapies is dramatically increased in 3 of the 4 MLMs. Lymphatic and/or lung metastasis occurs 100% of the time in one MLM line. This recurrent/metastatic model of HPV+ OPSCC retains the characteristics evident in refractory human disease (heterogeneity, resistance to therapy, metastasis in lymph nodes/lungs) thus serving as an ideal translational system to test novel therapeutics. Moreover, this system may provide insights into the molecular mechanisms of metastasis.

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

The behavior of the catanionic system of dioctadecyldimethylammonium bromide (DODAB) and sodium dodecyl sulfate (SDS) was investigated at 23 +/- 1 degrees C at the air-water interface using a Langmuir trough. The surface pressure as a function of surface area was measured while monitoring domain structures using epifluorescence microscopy. At high surface densities, the monolayer exhibits collapse through reversible folding at about 47 mN m(-1). This corresponds to the DODAB collapse surface pressure. The number of folds increases with the rate of compression speed and is history-dependent.

Effective treatments for recurrent/metastatic human papillomavirus-positive (HPV+) head and neck squamous cell cancer (HNSCC) are limited. To aid treatment development, we characterized a novel murine model of recurrent/metastatic HPV+ HNSCC. Further analysis of the parental tumor cell line and its four recurrent/metastatic derivatives led to preclinical testing of an effective treatment option for this otherwise fatal disease. Reverse phase protein arrays identified key signaling cascades in the parental and recurrent/metastatic cell lines. While protein expression profiles differed among the recurrent/metastatic cell lines, activated proteins associated with the mTOR signaling cascade were a commonality. Based on these data, mTOR inhibition was evaluated as an adjuvant treatment for recurrent/metastatic disease. mTOR activity and treatment response were assessed in vitro by western blot, Seahorse, proliferation, clonogenic, and migration assays. Standard-of-care cisplatin/radiation therapy (CRT) versus CRT/rapamycin were compared in vivo. Low-dose rapamycin inhibited mTOR signaling, decreasing proliferation (43%) and migration (62%) while it enhanced CRT-induced cytotoxicity (3.3 fold) in clonogenic assays. Furthermore, rapamycin re-sensitized CRT-resistant, metastatic tumors to treatment in vivo, improving long-term cures (0–30% improved to 78–100%, depending on the recurrent/metastatic cell line) and limiting lymph node metastasis (32%) and lung metastatic burden (30 fold). Studies using immune compromised mice suggested rapamycin's effect on metastasis is independent of the adaptive immune response. These data suggest a role of mTOR activation in HPV+ HNSCC recurrent/metastatic disease and that adjuvant mTOR inhibition may enhance treatment of resistant, metastatic cell populations at the primary site and limit distant metastasis.