Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.

The coordinated and dynamic regulation of adhesions is required for cell migration. We demonstrated previously that limited proteolysis of talin1 by the calcium-dependent protease calpain 2 plays a critical role in adhesion disassembly in fibroblasts ( The coordinated and dynamic regulation of adhesions is central for cell migration in both normal and pathological processes (1). Cell migration is initiated by forming protrusions, which are stabilized by integrin-mediated adhesions that establish structural and signaling linkages between the extracellular matrix and the actin cytoskeleton. Subsequently, actomyosin contractility drives forward propulsion, and finally, adhesions are disassembled to allow for directional movement. Thus, it is not surprising that efficient cell migration necessitates precise spatial and temporal control of these events. However, the mechanisms governing the formation and disassembly of adhesions are still not well understood.A prominent component involved in this regulation is focal adhesion kinase (FAK).2 FAK promotes cell migration by its capacity to orchestrate signals between integrin and growth factor receptors (2). Downstream of integrin or growth factor stimulation, FAK is phosphorylated at Tyr-397, which is an important binding site for Src family kinases (3). Previous studies have demonstrated a critical role for FAK as a regulator of adhesion dynamics (4 -7). In addition, FAK has been shown to mediate the tyrosine phosphorylation of p190RhoGAP as a negative regulator of Rho activity in focal adhesion turnover and polarity (5, 8). Furthermore, microtubule-induced focal adhesion disassembly requires FAK and dynamin (9). Nevertheless, the mechanisms by which FAK regulates the disassembly of focal adhesions remain to be elucidated. Evidence has emerged supporting the role of the calpain family of intracellular calcium-dependent proteases in regulating cell migration (10 -14). Calpains have been proposed to regulate migration, at least in part, through their ability to modulate the dynamics of adhesions (15). Numerous calpain targets have been identified, some of which are proteins that are present in focal adhesions, including talin, paxillin, and FAK (16). Thus, it is likely that proteolysis of these substrates contributes to the regulation of adhesion dynamics and cell migration.The involvement of both FAK and calpain in regulating the turnover of adhesions prompted us to investigate the cleavage of FAK by calpain as a possible mechanism by which FAK affects adhesion dynamics. We demonstrated previously that calpain-mediated proteolysis of talin regulates adhesion dynamics (17). Here, we show that FAK also regulates talin dynamics. We have identified the calpain cleavage site of FAK and have generated a mutant form of FAK that is resistant to calpain proteolysis. Expression of wild-type but not calpainresistant FAK restores the adhesion dynamics of talin in FAKdeficient cells. Taken together, our findings suggest a novel role for calpain-mediated cleavage of FAK in regulating...

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 cooperates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.