Shreya Mitra

Lactate Dehydrogenase B: A Metabolic Marker of Response to Neoadjuvant Chemotherapy in Breast Cancer

Purpose: Although breast cancers are known to be molecularly heterogeneous, their metabolic phenotype is less well-understood and may predict response to chemotherapy. This study aimed to evaluate metabolic genes as individual predictive biomarkers in breast cancer. Experimental Design: mRNA microarray data from breast cancer cell lines were used to identify bimodal genes—those with highest potential for robust high/low classification in clinical assays. Metabolic function was evaluated in vitro for the highest scoring metabolic gene, lactate dehydrogenase B (LDHB). Its expression was associated with neoadjuvant chemotherapy response and relapse within clinical and PAM50-derived subtypes. Results: LDHB was highly expressed in cell lines with glycolytic, basal-like phenotypes. Stable knockdown of LDHB in cell lines reduced glycolytic dependence, linking LDHB expression directly to metabolic function. Using patient datasets, LDHB was highly expressed in basal-like cancers and could predict basal-like subtype within clinical groups [OR = 21 for hormone receptor (HR)-positive/HER2-negative; OR = 10 for triple-negative]. Furthermore, high LDHB predicted pathologic complete response (pCR) to neoadjuvant chemotherapy for both HR-positive/HER2-negative (OR = 4.1, P < 0.001) and triple-negative (OR = 3.0, P = 0.003) cancers. For triple-negative tumors without pCR, high LDHB posttreatment also identified proliferative tumors with increased risk of recurrence (HR = 2.2, P = 0.006). Conclusions: Expression of LDHB predicted response to neoadjuvant chemotherapy within clinical subtypes independently of standard prognostic markers and PAM50 subtyping. These observations support prospective clinical evaluation of LDHB as a predictive marker of response for patients with breast cancer receiving neoadjuvant chemotherapy. Clin Cancer Res; 19(13); 3703–13. ©2013 AACR.

Rab25 increases cellular ATP and glycogen stores protecting cancer cells from bioenergetic stress

Cancer cells are metabolically stressed during tumour progression due to limited tumour vascularity and resultant nutrient, growth factor and oxygen deficiency that can induce cell death and inhibit tumour growth. We demonstrate that Rab25, a small GTPase involved in endosomal recycling, that is genomically amplified in multiple tumour lineages, is a key regulator of cellular bioenergetics and autophagy. RAB25 enhanced survival during nutrient stress by preventing apoptosis and autophagy via binding and activating AKT leading to increased glucose uptake and improved cellular bioenergetics. Unexpectedly, Rab25 induced the accumulation of glycogen in epithelial cancer cells, a process not previously identified. Strikingly, an increase in basal ATP levels combined with AKT‐dependent increases in glucose uptake and glycogen storage allowed maintenance of ATP levels during bioenergetic stress. The clinical relevance of these findings was validated by the ability of a Rab25‐dependent expression profile enriched for bioenergetics targets to identify patients with a poor prognosis. Thus, Rab25 is an unexpected regulator of cellular bioenergetics implicated as a useful biomarker and potential therapeutic target.

Rab25 in cancer: a brief update

Derailed endocytosis is a hallmark of cancer. The endocytic pathway, as demonstrated by our laboratory, is a frequent target of genomic aberrations in cancer and plays a critical role in the maintenance of cellular polarity, stem cell function, bioenergetics, proliferation, motility, invasion, metastasis, apoptosis and autophagy. The Rab GTPases, along with their effectors, are critical regulators of this endocytic machinery and can have a huge impact on the cellular itinerary of growth and metabolism. Rab25 is an epithelial-cell-specific member of the Rab GTPase superfamily, sharing close homology with Rab11a, the endosomal recycling Rab GTPase. RAB25 has been implicated in various cancers, with reports presenting it as both an oncogene and a tumour-suppressor gene. At the cellular level, Rab25 was shown to contribute to invasiveness of cancer cells by regulating integrin trafficking. Recently, our laboratory uncovered a critical role for Rab25 in cellular energetics. Assimilating all of the existing evidence, in the present review, we give an updated overview of the complex and often context-dependent role of Rab25 in cancer.

Interactions between tumor cells and microenvironment in breast cancer: A new opportunity for targeted therapy

Breast cancer remains the leading cause of morbidity and second‐leading cause of death in women. Despite efforts to uncover new targeted therapies, a vast number of women die due to refractory or recurrent breast tumors. Most breast cancer studies have focused on the intrinsic characteristics of breast tumor cells, including altered growth, proliferation, and metabolism. However, emerging research suggests that the tumor microenvironment can substantially affect relapse rates and therapeutic responses. In this review, we discuss the interactions between the tumor and microenvironment in breast cancer, with regard to mutational profiles and altered metabolism that could serve as potential therapeutic targets. We also describe current technologies available to study these interactions. (Cancer Sci 2012; 103: 400–407)

Rab25 acts as an oncogene in luminal B breast cancer and is causally associated with Snail driven EMT

The Rab GTPases regulate vesicular trafficking machinery that transports and delivers a diverse pool of cargo, including growth factor receptors, integrins, nutrient receptors and junction proteins to specific intracellular sites. The trafficking machinery is indeed a major posttranslational modifier and is critical for cellular homeostasis. Deregulation of this stringently controlled system leads to a wide spectrum of disorders including cancer. Herein we demonstrate that Rab25, a key GTPase, mostly decorating the apical recycling endosome, is a dichotomous variable in breast cancer cell lines with higher mRNA and protein expression in Estrogen Receptor positive (ER+ve) lines. Rab25 and its effector, Rab Coupling Protein (RCP) are frequently coamplified and coordinately elevated in ER+ve breast cancers. In contrast, Rab25 levels are decreased in basal-like and almost completely lost in claudin-low tumors. This dichotomy exists despite the presence of the 1q amplicon that hosts Rab25 across breast cancer subtypes and is likely due to differential methylation of the Rab25 promoter. Functionally, elevated levels of Rab25 drive major hallmarks of cancer including indefinite growth and metastasis but in case of luminal B breast cancer only. Importantly, in such ER+ve tumors, coexpression of Rab25 and its effector, RCP is significantly associated with a markedly worsened clinical outcome. Importantly, in claudin-low cell lines, exogenous Rab25 markedly inhibits cell migration. Similarly, during Snail-induced epithelial to mesenchymal transition (EMT) exogenous Rab25 potently reverses Snail-driven invasion. Overall, this study substantiates a striking context dependent role of Rab25 in breast cancer where Rab25 is amplified and enhances aggressiveness in luminal B cancers while in claudin-low tumors, Rab25 is lost indicating possible anti-tumor functions.

Rab11-FIP1C Is a Critical Negative Regulator in ErbB2-Mediated Mammary Tumor Progression.

Rab coupling protein (FIP1C), an effector of the Rab11 GTPases, including Rab25, is amplified and overexpressed in 10% to 25% of primary breast cancers and correlates with poor clinical outcome. Rab25 is also frequently silenced in triple-negative breast cancer, suggesting its ability to function as either an oncogene or a tumor suppressor, depending on the breast cancer subtype. However, the pathobiologic role of FIP family members, such as FIP1C, in a tumor-specific setting remains elusive. In this study, we used ErbB2 mouse models of human breast cancer to investigate FIP1C function in tumorigenesis. Doxycycline-induced expression of FIP1C in the MMTV-ErbB2 mouse model resulted in delayed mammary tumor progression. Conversely, targeted deletion of FIP1C in the mammary epithelium of an ErbB2 model coexpressing Cre recombinase led to accelerated tumor onset. Genetic and biochemical characterization of these FIP1C-proficient and -deficient tumor models revealed that FIP1C regulated E-cadherin (CDH1) trafficking and ZONAB (YBX3) function in Cdk4-mediated cell-cycle progression. Furthermore, we demonstrate that FIP1C promoted lysosomal degradation of ErbB2. Consistent with our findings in the mouse, the expression of FIP1C was inversely correlated with ErbB2 levels in breast cancer patients. Taken together, our findings indicate that FIP1C acts as a tumor suppressor in the context of ErbB2-positive breast cancer and may be therapeutically exploited as an alternative strategy for targeting aberrant ErbB2 expression. Cancer Res; 76(9); 2662-74. ©2016 AACR.

A murine preclinical syngeneic transplantation model for breast cancer precision medicine

This work presents a new preclinical platform for implementation of selected targeted therapeutics for breast cancer patients. We previously demonstrated that altered activity of lysophosphatidic acid in murine mammary glands promotes tumorigenesis. We have now established and characterized a heterogeneous collection of mouse-derived syngeneic transplants (MDSTs) as preclinical platforms for the assessment of personalized pharmacological therapies. Detailed molecular and phenotypic analyses revealed that MDSTs are the most heterogeneous group of genetically engineered mouse models (GEMMs) of breast cancer yet observed. Response of MDSTs to trametinib, a mitogen-activated protein kinase (MAPK) kinase inhibitor, correlated with RAS/MAPK signaling activity, as expected from studies in xenografts and clinical trials providing validation of the utility of the model. Sensitivity of MDSTs to talazoparib, a poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitor, was predicted by PARP1 protein levels and by a new PARP sensitivity predictor (PSP) score developed from integrated analysis of drug sensitivity data of human cell lines. PSP score–based classification of The Cancer Genome Atlas breast cancer suggested that a subset of patients with limited therapeutic options would be expected to benefit from PARP-targeted drugs. These results indicate that MDSTs are useful models for studies of targeted therapies, and propose novel potential biomarkers for identification of breast cancer patients likely to benefit from personalized pharmacological treatments.

Characterization of the Role Rab25 in Energy Metabolism and Cancer Using Extracellular Flux Analysis and Material Balance

Rab25, by altering trafficking of critical cellular resources, influences cell metabolism and survival during stress conditions. Overall, perturbations in the vesicular trafficking machinery change cellular bioenergetics that can be directly measured in real time as Oxygen Consumption Rate, OCR (mitochondrial respiration) and Extracellular Acidification Rate, ECAR (glycolysis) by an extracellular flux analyzer (XF96, Seahorse Biosciences, MA). Additionally, overall turnover of glucose, lactate, as well as glutamine and glutamate can be measured biochemically using the YSI2900 Biochemistry Analyzer (YSI Incorporated, Life Sciences, OH). A combination of these two methods allows a precise and quantitative approach to interrogate the role of Rab25 as well as other Rab GTPases in central carbon energy metabolism.

Abstract B40: Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25

Epithelial cells maintain intercellular communication directly by cell-cell, cell-ECM interaction and indirectly by paracrine secretion of extracellular messengers either as free molecules or via exosomes. The vesicular trafficking machinery is an integral part of such communication, paralleling digital cables that transmit the information bytes to the correct hub. During the process of EMT, derailing of the trafficking machinery leads to a systemic breakdown of intra and inter-cellular communication pathways. The purpose of the current study is to elucidate the functional role of Rab25, a key member of the vesicular trafficking machinery in mediating EMT-MET processes during breast cancer progression. Aberrant endocytosis is a new hallmark of cancer. Rab GTPases are the primary regulators of endocytosis. Many members of this large family are implicated in a wide array of cancers including breast cancer. Specifically, Rab25, a GTPase associated with the recycling endosomes, is deregulated in most epithelial cancers. Rab25 appears to function both as a tumor suppressor and an oncogene in a context dependent manner. We found that Rab25 is lost in Claudin Low tumors as well as in representative cell lines such as the MDA MB231. Rab25 is a robust and sufficient marker of breast cancers and cell lines with an increased EMT signature. Additionally, in immortalized human mammary epithelial cell lines (HMLE), induction of EMT by overexpression of Snail1 completely obliterates Rab25 protein. Exogenous re-expression of Rab25 dramatically rescues these cells from Snail driven invasion through Matrigel. However this Snail dependent regulation is unique only to TN and Claudin Low tumors and does not hold true for hormone receptor positive tumors. Further, analysis of RNA Seq data from a MMTV mouse model showed that tumors enriched in EMT markers and by corollary with low CDH and claudins, also lost Rab25 transcripts. Snail mediated transcriptional repression targets recycling endosomal proteins such as Rab25 to decrease delivery of junction proteins that facilitate cell-cell communication to the appropriate compartment. In parallel, it potentially enhances paracrine regulation by secreted exosomes. Overall, our study shows presence and absence of Rab25 markedly alters cell-cell communication, which is essential for maintenance of epithelial features of cells. Rab25 could be a prognostic marker and a therapeutic target for a subset of patients with increased risk of metastatic disease. Citation Format: Shreya Mitra, Lorenzo Federico, Gordon B. Mills. Transcription factor Snail mediates EMT by altering vesicular trafficking protein Rab25. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B40.

Abstract A17: TRPM7 kinase domain is involved in breast tumor cell metastasis

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Introduction: TRPM7 (transient receptor potential melastatin 7) is a non-selective cation channel fused to protein kinase domain at the C-terminal whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of breast cancer cells and promotes breast tumor metastasis. The lack of cell-permeable inhibitors of the kinase domain represents a barrier to understand the kinase function. Methods: Herein, we discover the first small molecule (KD-1) that targets TRPM7 kinase activity and characterize its mechanism of action in-vitro, in-cells and in-vivo. By using this inhibitor, we are investigated the involvement of TRPM7 kinase domain in breast tumor cell metastasis. Results: Mg2+ starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of Mg2+-starved HEK293 cells with KD-1 decreased eEF2 phosphorylation, consistent with TRPM7 kinase activity suppression in-cells. KD-1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA-MB-231 cells. And when MDA-MB-231 and BT 549 cells were treated with increasing doses of KD-1, no change in cell viability was seen. Interestingly, KD-1 inhibited migration and invasion in the MDA-MB-231 and BT 549 cells that is reportedly regulated by TRPM7 kinase activity. Finally, the bioluminescent signals (to assess lung metastasis) were significantly lower in KD-1-treated mice (25 and 50 mg/kg/day) than in mice treated with vehicle control (P ≤0.05, 2-sided t- test.). Conclusion: The discovered compound represents the first inhibitor that targets the kinase activity of transient receptor potential melastatin 7 (TRPM7). Inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis. Citation Format: Tamer S. Kaoud, Xuemei Xie, Jihyun Park, Clint D.J. Tavares, Shreya Mitra, Micael Cano, Mohamed F. Radwan, Chandra Bartholomeusz, Kevin N. Dalby. TRPM7 kinase domain is involved in breast tumor cell 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 A17.